Anti-Vista Antibodies And Fragments, Uses Thereof, And Methods Of Identifying Same

ABSTRACT

The present invention relates to antibodies and fragments that bind to a V-domain Ig Suppressor of T cell Activation (VISTA), and methods of eliciting certain biological responses using the antibodies. Compositions and methods of using anti-VISTA antibodies in combination with one or more antibodies that bind to immune checkpoint proteins are also provided.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/481,410, filed Apr. 6, 2017, now U.S. Pat. No. 11,014,987, which is aContinuation-in-part of U.S. patent application Ser. No. 15/107,784,filed Jun. 23, 2016, now U.S. Pat. No. 10,273,301, which is a U.S. Nat'lStage Appl. of Int'l Appl. No. PCT/IB2014/002868, filed Dec. 22, 2014,which claims the benefit of U.S. Provisional Appl. Nos. 62/319,605,filed Apr. 7, 2016, 62/085,086, filed Nov. 26, 2014, and 61/920,695,filed Dec. 24, 2013, each of which is hereby incorporated by referencein its entirety.

SEQUENCE DISCLOSURE

This application includes as part of its disclosure an electronicsequence listing text file named “1143260o105005.txt”, having a size of91,678 bytes and created on May 20, 2021, which is hereby incorporatedby reference in its entirety.

BACKGROUND OF THE INVENTION

The expression of negative immune regulators by cancer cells or immunecells in the tumor microenvironment can suppress the host's immuneresponse against the tumor. To effectively combat the cancer, it isdesirable to block tumor-mediated suppression of the host immuneresponse. Accordingly, there is a need for new and effective therapeuticagents that inhibit negative immune regulators in the tumormicroenvironment that suppress anti-tumor immune responses.

SUMMARY OF THE INVENTION

The present invention provides, in an embodiment, a pharmaceuticalcomposition comprising a) an antibody or antibody fragment thereofcomprising an antigen binding region that binds to a V-domain IgSuppressor of T cell Activation (VISTA); b) an antibody or antibodyfragment thereof comprising an antigen binding region that binds to animmune checkpoint protein; and c) a pharmaceutically acceptable carrier,diluent, or excipient.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising a) an antibody or antibody fragment thereofcomprising an antigen binding region that binds to VISTA; b) an antibodyor antibody fragment thereof comprising an antigen binding region thatbinds to a PD-1 protein, and c) a pharmaceutically acceptable carrier,diluent, or excipient.

In other embodiments, the present invention provides a method ofenhancing an immune response in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a) an antibody or an antibody fragment thereof comprising anantigen binding region that binds VISTA; and b) an antibody or antibodyfragment thereof comprising an antigen binding region that binds to animmune checkpoint protein, thereby enhancing an immune response to thecancer. In a particular embodiment, the immune checkpoint protein isPD-1. In certain embodiments, the individual in need thereof has cancer.

The compositions and methods of the present invention are useful for,e.g., enhancing immune responses (e.g., T cell responses, anti-tumorresponses) in individuals (e.g. humans) having cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A-1C: Graphs showing VISTA expression on TF1 AML Cells Expressionof VISTA protein by flow cytometry is shown in the TF-1 AML cell line.

FIG. 2A-2E: Graphs showing staining and gating strategies foridentification of Human Myeloid and Lymphoid Subsets.

FIG. 3A-3G: Graphs showing expression of VISTA on Human Myeloid andLymphoid Subsets from one healthy normal donor.

FIG. 4: Graph showing expression of VISTA on Human Myeloid and LymphoidSubsets across multiple healthy normal donors.

FIG. 5A-5B: Graph showing staining and gating strategies foridentification of expression of VISTA on Human Monocytes andMacrophages.

FIG. 6A-6C: Graphs showing expression of VISTA on Human Monocytes andMacrophages.

FIG. 7A-7E: Graphs showing staining and gating strategies foridentification of expression of VISTA on Human T and NK Cell Subsets.

FIG. 8A-8G: Graphs showing expression of VISTA on Human T and NK CellSubsets from one healthy normal donor.

FIG. 9: Graph showing expression of VISTA on Human T and NK Cell Subsetsacross multiple healthy normal donors.

FIG. 10A-10D: Graphs showing staining and gating strategies foridentification of expression of VISTA on Human Dendritic Cell subsets.

FIG. 11A-11C: Graphs showing expression of VISTA on Human Dendritic Cellsubsets and basophils from one healthy normal donor.

FIG. 12: Graph showing expression of VISTA on Human Dendritic CellSubsets and basophils across multiple healthy normal donors.

FIG. 13A-13D: Analysis of VISTA expression on healthy human peripheralblood cells. Profile of VISTA expression on healthy human peripheralblood cells using multicolor flow cytometry analysis: Whole bloodsamples from 2 different individuals were analyzed for VISTA expressionon (FIG. 13A) monocytesSS^(lo)′CD11b^(hi)CD14^(hi)CD16^(−ve)CD33^(+ve)HLA-DR^(+ve)CD19^(−ve))(FIG. 13B) neutrophils (SSC^(hi)CD177⁺CD11b^(hi)CD14^(lo)CD16^(+ve)CD33^(+ve)HLA-DR^(−ve)CD19^(−ve)). Peripheral bloodmononuclear cells were isolated using Ficoll gradient for analysis of(FIG. 13C) CD4+ T cells (CD3^(+ve)CD4^(+ve)), and (FIG. 13D) CD8+ Tcells (CD3^(+ve)CD8^(+ve)).

FIG. 14A-14C: Analysis of VISTA expression on peripheral blood cellsfrom a lung cancer patient and a healthy control donor. Profile of VISTAexpression on lung cancer patient peripheral blood cells usingmulticolor flow cytometry analysis: Representative FACS plot (FIG. 14A)from one individual is shown. Peripheral blood mononuclear cells wereisolated by Ficoll and analyzed for VISTA expression on (FIG. 14B)monocytes (CD14+CD11b+CD33+HLADR+CD15−) and (FIG. 14C) myeloid derivedsuppressor cells (CD14−CD11b+CD33−HLADR−CD15+CD16+).

FIG. 15A-15C: Profile of VISTA expression in peripheral blood cells froma patient with colon cancer, using multicolor flow cytometry analysis:Representative FACS plot (FIG. 15A) from one individual is shown.Peripheral blood mononuclear cells were isolated by Ficoll and analyzedfor VISTA expression on (FIG. 15B) monocytes(CD14+CD11b+CD33+HLADR+CD15−) and (FIG. 15C) myeloid derived suppressorcells (CD14−CD11b+CD33−HLADR-CD15+CD16+).

FIG. 16A-16D: Profile of VISTA expression on Cynomolgus monkeyperipheral blood cells using multicolor flow cytometry analysis: Wholeblood from 4 different monkeys was analyzed for VISTA expression on(FIG. 16A) monocytes (SSC^(lo)CD11b^(hi)CD14^(hi)HLA-DR^(hi)CD16^(−ve)CD19^(−ve) and (FIG. 16B) neutrophils CD11b^(hi)CD14^(lo)HLA-DR^(−ve)CD16^(−ve) CD19^(−ve). Peripheral blood mononuclear cellsfrom three monkeys were isolated using Ficoll gradient for analysis of(FIG. 16C) CD4+ T cells (TCRα/β^(+ve)CD4^(+ve)) and (FIG. 16D) CD8+ Tcells (TCRα/β^(+ve)CD8^(+ve))

FIG. 17: Graph showing absolute expression values of VISTA RNA in Hemecell lines.

FIG. 18: Mouse A20 cells were stably transfected with either GFP orhuman VISTA. They were incubated with ova peptide and with DO11.10 Tcells. CD25 expression by the T cells was measured 24 hours afterincubation began. The A20-huVISTA cells suppress CD25 expression by theT cells, but this readout is significantly restored by incubation withVSTB95.

FIG. 19A-19F: Graphs showing Human VISTA ELISA results.

FIG. 20A-20F: Human VISTA FACS results, showing anti-VISTA antibodiesbinding to cells expressing human VISTA.

FIG. 21A-21D: Dilution study of 6 anti-VISTA antibody candidates in themixed lymphocyte reaction from 30 μg/ml to 0.0 μg/ml.

FIG. 22A-22B: Dilution studies of 6 anti-VISTA antibody candidates inthe SEB assay (individual CPM counts and IFN-g concentrations) from 30μg/ml to 0.0 μg/ml.

FIG. 23: Sensorgram plot using anti-VISTA antibody VSTB85 coated on aProteon SPR chip and VISTA protein with the indicated competitors runover the chip (competitors listed in Table 16).

FIG. 24: Experimental design for MB49 murine bladder tumor model

FIG. 25A-25B: MB49 tumor growth in female C57Bl/6 mice. Graphsillustrate tumor growth in individual mice treated with anti-mouse VISTAantibody (FIG. 25B) or control IgG (FIG. 25A).

FIG. 26: Amino acid sequence of human VISTA (SEQ ID NO:46).

FIG. 27: Multiple sequence alignment of VISTA orthologues

FIG. 28: Regions of human VISTA bound by VSTB50 and VSTB60 antibodies(top) or VSTB95 and VSTB112 antibodies (bottom), as determined by HDX

FIG. 29: VISTA Epitope bound by VSTB112. (Top) VISTA is shown in cartoonwith strands labeled. Residues having at least one atom within 5 Å ofVSTB112 in the complex are colored blue. Blue and orange sphereshighlight a chain break, and the cyan and green spheres mark the N- andC-termini of the VISTA structure, respectively. (Bottom) Sequence ofVISTA construct used in structure determination. Circles below thesequence are used to indicate residues which make only main chaincontacts with VSTB112, triangles indicate a side chain contact, andsquares indicate the side chain contact results in either a hydrogenbond or salt bridge interaction as calculated by PISA. Shapes arecolored to indicate the CDR having the greatest number of atomscontacted by the given residue with CDR colors defined in FIG. 59.Secondary structural elements are as defined in the program MOE withyellow arrows representing β-strands and red rectangles indicatingα-helices.

FIG. 30: VSTB112 Paratope. (Top) VISTA antigen is shown in illustrationand VSTB112 within 5 angstrom (Å) of VISTA is shown in surface withcolors used to designate CDR identity as specified in the sequencebelow. Contacting framework residues adjacent to a CDR are coloredsimilarly to the corresponding CDR (Bottom) Sequence of VSTB112 Fvregion. Colored backgrounds specify CDRs according to Kabat definitions.Circles below the sequence are used to indicate residues which make mainchain only contacts with VISTA, triangles indicate a side-chain contact,and squares indicate the side chain contact results in either a hydrogenbond or salt bridge interaction as calculated by PISA.

FIG. 31: Comparison of epitope regions identified by crystallography andhydrogen deuterium exchange (HDX). Sequence of VISTA construct used instructure determination. Circles below the sequence are used to indicateresidues which make only main chain contacts with VSTB112, trianglesindicate a side chain contact, and squares indicate the side chaincontact results in either a hydrogen bond or salt bridge interaction ascalculated by PISA.

FIG. 32: Activation of CD14+ monocytes in whole PBMC by VSTB174 (derivedfrom VSTB112). In each part of the experiment, cells were incubated withPBS, IgG1 control antibody, or VSTB174 at 1, 0.1 or 0.01 ug/ml. Leftpanel shows CD80 MFI; right panel shows HLA-DR MFI (two donors testedwith representative results shown).

FIG. 33: Graph showing ADCC activity of VSTB174 directed againstK562-VISTA cells.

FIG. 34: Graph showing ADCP activity of VSTB174 directed againstK562-VISTA cells. Both antibodies depicted have the same Fab, butVSTB174 has an IgG1 Fc and VSTB140 has Fc silent IgG2.

FIG. 35: Graph showing phagocytosis mediated by VSTB174, VSTB149 orVSTB140 mAbs against K562-VISTA. Each mAb was tested with 7 half logdoses, ranging from 0.0008 μg/ml to 0.56 ug/ml.

FIG. 36: Graph showing phagocytosis mediated by VSTB174, VSTB149 orVSTB140 mAbs against myeloma cell line K562 cells. Each mAb was testedwith 7 half log doses, ranging from 0.0008 μg/ml to 0.56 ug/ml.

FIG. 37: MB49 tumor efficacy study evaluating VSTB123 1, 5, 7.5, and 10mg/kg in female VISTA-KI mice. Tumor volumes were approximately 50 mm³when dosing began at day 6 after implant. VSTB123 is the VSTB112 Fabgrafted onto a mouse Fc scaffold and binds to human VISTA in theVISTA-KI mouse.

FIG. 38: Graph shows that CD14+ cells expressing high/intermediatelevels of VISTA are found in 13/13 lung cancer samples, as well as indistant lung tissue and peripheral blood of patients.

FIG. 39: IHC staining for VISTA in Lung Cancer using GG8.

FIG. 40: Anti-VISTA antibody triggers monocyte activation via CD16crosslinking. PD-L1 expression is used as a marker of myeloid activationusing human PBMCs. Anti-CD16, used as the positive control, inducedrobust monocyte activation; Fc block (mix of Fc IgG1 fragments), used asthe negative control, blocked monocyte activation. VSTB112, but notVSTB140, induces monocyte activation compared to the human IgG1 control.

FIG. 41: Schematic of study design for VSTB123 or VSTB124 effect on thegrowth of established MB49 tumors in hVISTA KI (knock-in) mice.Antibodies were injected on study days 5, 7, 10, 12, 14, 17, 19, 21, 24,and 26. Blood samples were collected on days −2, −1, 0, 4, 7, 11, 14,18, 24, 31, and 39. If blood samples were taken on the same day as anantibody injection, blood samples were taken first.

FIGS. 42A and 42B: MB49 tumor growth in hVISTA KI female mice (FIG. 42A)and survival of mice bearing MB49 tumors after treatment with VSTB123 orVSTB124 in hVISTA KI female mice (FIG. 42B). In FIG. 42A, mean tumorvolume measurements (mm³) are shown for female mice treated with VSTB123or VSTB124 as compared to the mouse IgG2a control group. The treatmentperiod was from day 5-26. Mean+/−SEM shown. Note that the y axes differamong the graphs. Data for the female mice are graphed until day 33,when >70% of mice were still alive in each group (n=6 or 7 per group).In FIG. 42B, the percent survival of hVISTA KI female mice is graphed,VSTB123 10 mg/kg, p=0.0108. The treatment period was from day 5-26.

FIG. 43: Fold-change in expression of 41 cytokines by PBMC treated withVSTB174. Whole PBMC from three healthy human donors were treated withVSTB174 or IgG1 control antibodies for 24 hours at the indicatedconcentrations. Cytokine production was analyzed by a 41-cytokinemultiplex kit. Average fold-change in expression over the IgG1 controlis shown as a heat map with a log color scale. Asterisks indicatesignificant differences between treatment and control sample means.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Out-of-range (OOR) denotesthat all samples for that cytokine in that donor were beyond the limitsof accurate detection (>above, <below).

FIGS. 44A and 44B: Macrophage activation in MB49 tumors. FIG. 44A showsa schematic of the study design. In FIG. 44B indicates increased CD80+macrophages in the tumor microenvironment with VSTB123, but not VSTB124.MB49 tumor-bearing hVISTA KI mice were treated with VSTB123, VSTB124, orcontrol mIgG2a and their tumors were analyzed, 24 h post-third dose, todetermine the relative expression of CD80 on tumor-infiltratingmacrophages. Each group contained 5 mice. Horizontal bars indicatemeans. *p<0.05.

FIG. 45: Migration of MPO+ cells to the tumor microenvironment.

FIG. 46: VSTB174 induces transient decrease in neutrophils.

FIG. 47: Anti-VISTA antibody (e.g., VSTB174) proposed mechanism ofaction.

FIG. 48: Expression profile of activation markers on immune cells, asindicated (CD80 on monocytes; CD69+ on CD8 T and NK cells; CD25+ on CD4T cells) at 24 hours after treatment, as indicated (e.g., VSTB174 orVSTB140 or corresponding controls).

FIG. 49: Myeloid depletion study design—treatment and analysis scheduleschematic. Mice were injected with MB49 tumor cells on day 0.Administration of VSTB123 or mouse IgG2a (mIgG2a) occurred on day 7, 9and 11 (downward-pointing arrows). Anti-GR1 antibody was administeredfor 7 doses EOD (day 5, 7, 9, 11, 13, 15 and 17) to deplete monocytesand granulocytes. Clodronate liposomes were administered on day 4, 10and 16 to deplete macrophages and dendritic cells. All antibodies andtherapies were injected via intraperitoneal route. Blood was drawn ondays 7 and 22 to evaluate immune cell depletion.

FIG. 50: Effects of macrophage (left), CD4+ T cell (middle), or CD8+ Tcell (right) depletion on efficacy of VSTB123 in MB49 bladder carcinomamodel.

FIG. 51: T cell and NK cell depletion study design treatment andanalysis schedule schematic. Mice were injected with MB49 tumor cells onday 0, and randomized among the groups on day 4. Therapeutic antibodyadministration (VSTB123 or mIgG2a control) occurred on days 7, 9 and 11.Depleting antibodies were administered on days 5, 7, 12, 17, 22, and 27.Blood was collected from 10 mice for baseline evaluation on day −4 andfrom 5 mice/group on day 7 prior to re-dosing with depleting antibodies.N=10 mice/group.

FIG. 52: Anti-VISTA and anti-PD-1 combination study design.FFPE=formalin fixed paraffin embedded; ICS=intracellular cytokinestaining.

FIG. 53: Synergistic effect of anti-VISTA antibody and anti-PD-1antibody (RMP1-14) on tumor growth inhibition and survival on MB49bladder carcinoma in male VISTA KI mice.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The present invention relates to antibodies to novel Immunoglobulinfamily ligand designated V-domain Immunoglobulin Suppressor of T cellActivation (VISTA) (Genbank: JN602184) (Wang et al., 2010, 2011). VISTAbears homology to PD-L1 but displays a unique expression pattern that isrestricted to the hematopoietic compartment. Specifically, VISTA isconstitutively and highly expressed on CD11b^(high) myeloid cells, andexpressed at lower levels on CD4⁺ and CD8⁺ T cells. The human homologueshares approximately 85% homology with murine VISTA and has similarexpression patterns (Lines et al., Cancer Research 74:1924, 2014). VISTAexpressed on antigen presenting cells (APCs) suppresses CD4⁺ and CD8⁺ Tcell proliferation and cytokine production via a cognate receptorindependent of PD-1. In a passive EAE (experimental autoimmuneencephalomyelitis) disease model, a VISTA specific monoclonal antibodyenhanced T-cell dependent immune responses and exacerbated disease.VISTA over-expression on tumor cells impaired protective anti-tumorimmunity in tumor-bearing hosts. Studies of human VISTA confirmed itssuppressive function on human T cells (Lines et al Cancer Research74:1924, 2014. Studies from Flies et al. also identified VISTA (namedPD-1H) as a potent immune suppressive molecule (Flies et al., 2011).VISTA is described in further detail in U.S. Published application US20130177557 A1 and U.S. Pat. Nos. 7,919,585 and 8,236,304, all of whichare incorporated herein by reference in their entirety.

VISTA is a novel negative immune regulator that suppresses immuneresponses. As described, for example, in Example 12 herein, treatmentwith a VISTA-specific monoclonal antibody in murine tumor models hasbeen shown to reverse the suppressive character of the tumor immunemicroenvironment and enhance protective anti-tumor immunity, thus,demonstrating the potential of a VISTA monoclonal antibody as a noveltherapeutic for cancer immunotherapy.

Antibodies and Fragments of the Present Invention

The term “antibody” is meant to include polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies,human antibodies and anti-idiotypic (anti-Id) antibodies, as well asfragments, regions or derivatives thereof, provided by any knowntechnique, such as, but not limited to, enzymatic cleavage, peptidesynthesis or recombinant techniques. Anti-VISTA antibodies of thepresent invention are capable of binding portions of VISTA thatmodulate, regulate, or enhance an immune response. In some embodiments,the antibodies competitively inhibit one or more of the anti-VISTAantibodies described herein. Methods for determining whether two or moreantibodies compete for binding to the same target are known in the art.For example, a competitive binding assay can be used to determinewhether one antibody blocks the binding of another antibody to thetarget. Typically, a competitive binding assay involves the use ofpurified target antigen (e.g., PD-1) bound either to a solid substrateor cells, an unlabeled test binding molecule, and a labeled referencebinding molecule. Competitive inhibition is measured by determining theamount of label bound to the solid surface or cells in the presence ofthe test binding molecule. Usually the test binding molecule is presentin excess. Typically, when a competing binding molecule is present inexcess, it will inhibit specific binding of a reference binding moleculeto a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%,or more. In some embodiments, competitive inhibition is determined usinga competitive inhibition ELISA assay.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen. Amonoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which population contains substantiallysimilar epitope binding sites. Monoclonal antibodies may be obtained bymethods known to those skilled in the art. See, for example Kohler andMilstein, Nature, 256:495-497 (1975); U.S. Pat. No. 4,376,110; Ausubelet al., eds., Current Protocols in Molecular Biology, Greene PublishingAssoc. and Wiley Interscience, N.Y., (1987, 1992); and Harlow and LaneANTIBODIES: A Laboratory Manual Cold Spring Harbor Laboratory (1988);Colligan et al., eds., Current Protocols in Immunology, GreenePublishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), thecontents of all of which are incorporated entirely herein by reference.Such antibodies may be of any immunoglobulin class including IgG, IgM,IgE, IgA, GILD and any subclass thereof. A hybridoma producing amonoclonal antibody of the present invention may be cultivated in vitro,in situ or in vivo.

The invention also encompasses digestion fragments, specified portionsand variants thereof, including antibody mimetics or comprising portionsof antibodies that mimic the structure and/or function of an antibody orspecified fragment or portion thereof, including single chain antibodiesand fragments thereof. Functional fragments include antigen-bindingfragments that bind to a mammalian VISTA protein. For example, antibodyfragments capable of binding to VISTA or portions thereof, including,but not limited to Fab (e.g., by papain digestion), Fab′ (e.g., bypepsin digestion and partial reduction) and F(ab′)₂ (e.g., by pepsindigestion), facb (e.g., by plasmin digestion), pFc′ (e.g., by pepsin orplasmin digestion), Fd (e.g., by pepsin digestion, partial reduction andreaggregation), Fv or scFv (e.g., by molecular biology techniques)fragments, are encompassed by the invention (see, e.g., Colligan,Immunology, supra). Antibody fragments of the present invention alsoinclude those discussed and described in Aaron L. Nelson, mAbs 2:1,77-83 (January/February 2010), the contents of which are incorporated byreference in their entirety.

Such fragments can be produced, for example, by enzymatic cleavage,synthetic or recombinant techniques, as known in the art and/or asdescribed herein. antibodies can also be produced in a variety oftruncated forms using antibody genes in which one or more stop codonshave been introduced upstream of the natural stop site. For example, acombination gene encoding a F(ab′)₂ heavy chain portion can be designedto include DNA sequences encoding the CH1 domain and/or hinge region ofthe heavy chain. The various portions of antibodies can be joinedtogether chemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

In one embodiment, the amino acid sequence of an immunoglobulin chain,or portion thereof (e.g., variable region, CDR) has about 70-100%identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 orany range or value therein) to the amino acid sequence of thecorresponding variable sequence chain described herein. Preferably,70-100% amino acid identity (e.g., 85, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100 or any range or value therein) is determined using asuitable computer algorithm, as known in the art.

Examples of heavy chain and light chain variable regions sequences areprovided herein.

The antibodies of the present invention, or specified variants thereof,can comprise any number of contiguous amino acid residues from anantibody of the present invention, wherein that number is selected fromthe group of integers consisting of from 10-100% of the number ofcontiguous residues in an anti-TNF antibody. Optionally, thissubsequence of contiguous amino acids is at least about 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250 or more amino acids in length, or any rangeor value therein. Further, the number of such subsequences can be anyinteger selected from the group consisting of from 1 to 20, such as atleast 2, 3, 4, or 5.

As those of skill will appreciate, the present invention includes atleast one biologically active antibody of the present invention.Biologically active antibodies have a specific activity at least 20%,30%, or 40%, and preferably at least 50%, 60%, or 70%, and mostpreferably at least 80%, 90%, or 95%-100% of that of the native(non-synthetic), endogenous or related and known antibody. Methods ofassaying and quantifying measures of enzymatic activity and substratespecificity, are well known to those of skill in the art.

Substantial similarity refers to a compound having at least 85% (e.g.,at least 95%) identity and at least 85% (e.g., at least 95%) of activityof the native (non-synthetic), endogenous or related and known antibody.

As used herein, the term “human antibody” refers to an antibody in whichsubstantially every part of the protein (e.g., CDR, framework, CL, CHdomains (e.g., CH1, CH2, CH3), hinge, (VL, VH)) is substantiallynon-immunogenic in humans, with only minor sequence changes orvariations. Similarly, antibodies designated primate (monkey, baboon,chimpanzee, and the like), rodent (mouse, rat, and the like) and othermammals designate such species, sub-genus, genus, sub-family, familyspecific antibodies. Further, chimeric antibodies can include anycombination of the above. Such changes or variations optionally andpreferably retain or reduce the immunogenicity in humans or otherspecies relative to non-modified antibodies. Thus, a human antibody isdistinct from a chimeric or humanized antibody. It is pointed out that ahuman antibody can be produced by a non-human animal or prokaryotic oreukaryotic cell that is capable of expressing functionally rearrangedhuman immunoglobulin (e.g., heavy chain and/or light chain) genes.Further, when a human antibody is a single chain antibody, it cancomprise a linker peptide that is not found in native human antibodies.For example, an Fv can comprise a linker peptide, such as two to abouteight glycine or other amino acid residues, which connects the variableregion of the heavy chain and the variable region of the light chain.Such linker peptides are considered to be of human origin.

Bispecific, heterospecific, heteroconjugate or similar antibodies canalso be used that are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forat least one VISTA protein, the other one is for any other antigen.Methods for making bispecific antibodies are known in the art. Therecombinant production of bispecific antibodies can be based on theco-expression of two immunoglobulin heavy chain-light chain pairs, wherethe two heavy chains have different specificities (Milstein and Cuello,Nature 305:537 (1983)). See also WO 93/08829, U.S. Pat. Nos. 6,210,668,6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453, 6,010,902,5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985, 5,821,333,5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549, 4,676,980, WO91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBO J. 10:3655(1991), Suresh et al., Methods in Enzymology 121:210 (1986), eachentirely incorporated herein by reference.

In one embodiment, the invention relates to a bispecific antibodytargeting VISTA and a second target protein (e.g., an immune checkpointprotein). Exemplary bispecific antibodies include a bispecific antibodytargeting VISTA and PD-L1 and a bispecific antibody targeting VISTA andPD-L2.

Human antibodies that are specific for human VISTA proteins or fragmentsthereof can be raised against an appropriate immunogenic antigen, suchas VISTA protein or a portion thereof (including synthetic molecules,such as synthetic peptides).

Other specific or general mammalian antibodies can be similarly raised.Immunogenic antigens preparation and monoclonal antibody production canbe performed using any suitable technique.

For example, a hybridoma is produced by fusing a suitable immortal cellline (e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or thelike, or heteromylomas, fusion products thereof, or any cell or fusioncell derived therefrom, or any other suitable cell line as known in theart, See, e.g., www.atcc.org, with antibody-producing cells.Antibody-producing cells can include isolated or cloned spleen,peripheral blood, lymph, tonsil, or other immune cells (e.g., B cells),or any other cells expressing heavy or light chain constant or variableor framework or complementarity determining region (CDR) sequences. Suchantibody-producing cells can be recombinant or endogenous cells, and canalso be prokaryotic or eukaryotic (e.g., mammalian, such as, rodent,equine, ovine, goat, sheep, primate). See, e.g., Ausubel, supra, andColligan, Immunology, supra, chapter 2, entirely incorporated herein byreference.

Antibody producing cells can also be obtained from the peripheral bloodor, the spleen or lymph nodes, of humans or other suitable animals thathave been immunized with the antigen of interest. Any other suitablehost cell can also be used for expressing heterologous or endogenousnucleic acid encoding an antibody, specified fragment or variantthereof, of the present invention. Fused cells (hybridomas) orrecombinant cells can be isolated using selective culture conditions orother suitable known methods, and cloned by limiting dilution or cellsorting, or other known methods. Cells which produce antibodies with thedesired specificity can be selected by a suitable assay (e.g.,enzyme-linked immunosorbent assay (ELISA)).

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from Cambridge antibody Technologies, Cambridgeshire, UK;MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK;Bioinvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma,Berkeley, Calif.; Ixsys. See, e.g., PCT/GB91/01134; PCT/GB92/01755;PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; PCT/GB94/01422;PCT/GB94/02662; PCT/GB97/01835; WO90/14443; WO90/14424; WO90/14430;PCT/US94/1234; WO92/18619; WO96/07754; EP 614 989; WO95/16027;WO88/06630; WO90/3809; U.S. Pat. No. 4,704,692; PCT/US91/02989;WO89/06283; EP 371 998; EP 550 400; EP 229 046; PCT/US91/07149; orstochastically-generated peptides or proteins—U.S. Pat. Nos. 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862;WO 86/05803, EP590 689, each entirely incorporated herein by reference, or that relyupon immunization of transgenic animals (e.g., SCID mice, Nguyen et al.,Microbiol. Immunol. 41:901-907 (1997); Sandhu et al., Crit. Rev.Biotechnol. 16:95-118 (1996); Eren et al., Immunol. 93:154-161 (1998),each entirely incorporated by reference as well as related patents andapplications) that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal., Proc. Natl. Acad. Sci. USA, 94:4937-4942 (May 1997); Hanes et al.,Proc. Natl. Acad. Sci. USA, 95:14130-14135 (November 1998)); single cellantibody producing technologies (U.S. Pat. No. 5,627,052, Wen et al., J.Immunol. 17:887-892 (1987); Babcook et al., Proc. Natl. Acad. Sci. USA93:7843-7848 (1996)); gel microdroplet and flow cytometry (Powell etal., Biotechnol. 8:333-337 (1990); One Cell Systems, Cambridge, Mass.;Gray et al., J. Imm. Meth. 182:155-163 (1995); Kenny et al.,Bio/Technol. 13:787-790 (1995)); B-cell selection (Steenbakkers et al.,Molec. Biol. Reports 19:125-134 (1994); Jonak et al., Progress Biotech,Vol. 5, In Vitro Immunization in Hybridoma Technology, Borrebaeck, ed.,Elsevier Science Publishers B.V., Amsterdam, Netherlands (1988)).

Methods for engineering or humanizing non-human or human antibodies canalso be used and are well known in the art. Generally, a humanized orengineered antibody has one or more amino acid residues from a sourcewhich is non-human, e.g., but not limited to mouse, rat, rabbit,non-human primate or other mammal. These human amino acid residues areoften referred to as “import” residues, which are typically taken froman “import” variable, constant or other domain of a known humansequence. Known human Ig sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.atcc.org/phage/hdb.html,each entirely incorporated herein by reference.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, avidity, specificity, half-life, orany other suitable characteristic, as known in the art. Generally partor all of the non-human or human CDR sequences are maintained while partor all of the non-human sequences of the framework and/or constantregions are replaced with human or other amino acids. Antibodies canalso optionally be humanized with retention of high affinity for theantigen and other favorable biological properties usingthree-dimensional immunoglobulin models that are known to those skilledin the art. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, framework (FR) residues can be selected andcombined from the consensus and import sequences so that the desiredantibody characteristic, such as increased affinity for the targetantigen(s), is achieved. In general, the CDR residues are directly andmost substantially involved in influencing antigen binding. Humanizationor engineering of antibodies of the present invention can be performedusing any known method, such as but not limited to those described in,for example, Winter (Jones et al., Nature 321:522 (1986); Riechmann etal., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)),Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol.Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A.89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat.Nos. 5,723,323, 5,976,862, 5,824514, 5,817483, 5,814476, 5,763,192,5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762,5,530,101, 5,585,089, 5,225,539; 4,816,567, each entirely incorporatedherein by reference, included references cited therein.

The anti-VISTA antibody can also be optionally generated by immunizationof a transgenic animal (e.g., mouse, rat, rabbit, hamster, non-humanprimate, and the like) capable of producing a repertoire of humanantibodies, as described herein and/or as known in the art. Cells thatproduce a human anti-VISTA antibody can be isolated from such animalsand immortalized using suitable methods, such as the methods describedherein.

Transgenic animals that can produce a repertoire of human antibodiesthat bind to human antigens can be produced by known methods (e.g., butnot limited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806,5,625,126, 5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued toLonberg et al.; Jakobovits et al. WO 98/50433, Jakobovits et al. WO98/24893, Lonberg et al. WO 98/24884, Lonberg et al. WO 97/13852,Lonberg et al. WO 94/25585, Kucherlapate et al. WO 96/34096,Kucherlapate et al. EP 0463 151 Bl, Kucherlapate et al. EP 0710 719 A1,Surani et al. U.S. Pat. No. 5,545,807, Bruggemann et al. WO 90/04036,Bruggemann et al. EP 0438 474 Bl, Lonberg et al. EP 0814 259 A2, Lonberget al. GB 2 272 440 A, Lonberg et al. Nature 368:856-859 (1994), Tayloret al., Int. Immunol. 6(4)579-591 (1994), Green et al, Nature Genetics7:13-21 (1994), Mendez et al., Nature Genetics 15:146-156 (1997), Tayloret al., Nucleic Acids Research 20(23):6287-6295 (1992), Tuaillon et al.,Proc Natl Acad Sci USA 90(8)3720-3724 (1993), Lonberg et al., Int RevImmunol 13(1):65-93 (1995) and Fishwald et al., Nat Biotechnol14(7):845-851 (1996), which are each entirely incorporated herein byreference). Generally, these mice comprise at least one transgenecomprising DNA from at least one human immunoglobulin locus that isfunctionally rearranged, or which can undergo functional rearrangement.The endogenous immunoglobulin loci in such mice can be disrupted ordeleted to eliminate the capacity of the animal to produce antibodiesencoded by endogenous genes.

Screening antibodies for specific binding to similar proteins orfragments can be conveniently achieved using peptide display libraries.This method involves the screening of large collections of peptides forindividual members having the desired function or structure. Antibodyscreening of peptide display libraries is well known in the art. Thedisplayed peptide sequences can be from 3 to 5000 or more amino acids inlength, frequently from 5-100 amino acids long, and often from about 8to 25 amino acids long. In addition to direct chemical synthetic methodsfor generating peptide libraries, several recombinant DNA methods havebeen described. One type involves the display of a peptide sequence onthe surface of a bacteriophage or cell. Each bacteriophage or cellcontains the nucleotide sequence encoding the particular displayedpeptide sequence. Such methods are described in PCT Patent PublicationNos. 91/17271, 91/18980, 91/19818, and 93/08278. Other systems forgenerating libraries of peptides have aspects of both in vitro chemicalsynthesis and recombinant methods. See, PCT Patent Publication Nos.92/05258, 92/14843, and 96/19256. See also, U.S. Pat. Nos. 5,658,754;and 5,643,768. Peptide display libraries, vector, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.), and Cambridge antibody Technologies (Cambridgeshire, UK). See,e.g., U.S. Pat. Nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203,5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260,5,856,456; 5,223,409, 5,403,484, 5,571,698, 5,837,500, assigned to Dyax,U.S. Pat. Nos. 5,427,908, 5,580,717; 5,885,793, assigned to Cambridgeantibody Technologies; U.S. Pat. No. 5,750,373, assigned to Genentech,U.S. Pat. Nos. 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493,and 5,698,417.

Antibodies of the present invention can also be prepared using at leastone anti-VISTA antibody encoding nucleic acid to provide transgenicanimals, such as goats, cows, sheep, and the like, that produce suchantibodies in their milk. Such animals can be provided using knownmethods. See, e.g., but not limited to, U.S. Pat. Nos. 5,827,690;5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489, andthe like, each of which is entirely incorporated herein by reference.

The anti-VISTA antibodies of the present invention can also be producedusing transgenic plants, according to known methods. See also, e.g.,Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October, 1999),Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) andreferences cited therein; Ma et al., Trends Biotechnol. 13:522-7 (1995);Ma et al., Plant Physiol. 109:341-6 (1995); Whitelam et al., Biochem.Soc. Trans. 22:940-944 (1994); and references cited therein. Each of theabove references is entirely incorporated herein by reference.

The antibodies of the invention can bind human VISTA with a wide rangeof affinities (K_(D)). In a preferred embodiment, at least one humanmonoclonal antibody of the present invention can optionally bind humanVISTA with high affinity. For example, a human monoclonal antibody canbind human VISTA with a K_(D) equal to or less than about 10⁻⁷ M, suchas but not limited to, 0.1-9.9 (or any range or value therein)×10⁻⁷,10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ or any range or value therein. Insome embodiments, the antibody or antibody fragment can binds humanVISTA with an affinity of at least 1×10⁻⁷ liter/mole, for example, atleast 1×10⁻⁸ liter/mole, for example, at least 1×10⁻⁹ liter/moleliter/mole.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method. (See, for example, Berzofsky,et al., “Antibody-Antigen Interactions,” In Fundamental Immunology,Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, JanisImmunology, W.H. Freeman and Company: New York, N.Y. (1992); and methodsdescribed herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters (e.g., K_(D), K_(a),K_(d)) are preferably made with standardized solutions of antibody andantigen, and a standardized buffer.

Nucleic Acid Molecules

Using the information provided herein, such as the nucleotide sequencesencoding at least 70-100% of the contiguous amino acids of at least oneof specified fragments, variants or consensus sequences thereof, or adeposited vector comprising at least one of these sequences, a nucleicacid molecule of the present invention encoding at least one anti-VISTAantibody comprising all of the heavy chain variable CDR regions of SEQID NOS:1, 2 and 3 and/or all of the light chain variable CDR regions ofSEQ ID NOS:4, 5 and 6 can be obtained using methods described herein oras known in the art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF), forexample, but not limited to, at least one specified portion of at leastone CDR, as CDR1, CDR2 and/or CDR3 of at least one heavy chain or lightchain; nucleic acid molecules comprising the coding sequence for ananti-VISTA antibody or fragment, e.g., a fragment comprising a variableregion; and nucleic acid molecules which comprise a nucleotide sequencedifferent from those described above but which, due to the degeneracy ofthe genetic code, still encode at least one anti-VISTA antibody asdescribed herein and/or as known in the art. It would be routine for oneskilled in the art to generate such degenerate nucleic acid variantsthat code for specific anti-VISTA antibodies of the present invention.See, e.g., Ausubel, et al., supra, and such nucleic acid variants areincluded in the present invention.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding an anti-VISTA antibody caninclude, but are not limited to, those encoding the amino acid sequenceof an antibody fragment; the coding sequence for the entire antibody ora portion thereof; the coding sequence for an antibody, fragment orportion, as well as additional sequences, such as the coding sequence ofat least one signal leader or fusion peptide, with or without theaforementioned additional coding sequences, such as at least one intron,together with additional, non-coding sequences, including but notlimited to, non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals (forexample—ribosome binding and stability of mRNA); an additional codingsequence that codes for additional amino acids, such as those thatprovide additional functionalities. Thus, the sequence encoding anantibody can be fused to a marker sequence, such as a sequence encodinga peptide that facilitates purification of the fused antibody comprisingan antibody fragment or portion.

Human genes which encode the constant (C) regions of the antibodies,fragments and regions of the present invention can be derived from ahuman fetal liver library, by known methods. Human C regions genes canbe derived from any human cell including those which express and producehuman immunoglobulins. The human C_(H) region can be derived from any ofthe known classes or isotypes of human H chains, including γ, μ, α, δ orε and subtypes thereof, such as G1, G2, G3 and G4. Since the H chainisotype is responsible for the various effector functions of anantibody, the choice of C_(H) region will be guided by the desiredeffector functions, such as complement fixation, or activity inantibody-dependent cellular cytotoxicity (ADCC).

Compositions

The pharmaceutical compositions disclosed herein are prepared inaccordance with standard procedures and are administered at dosages thatare selected to treat, e.g., reduce, prevent, or eliminate, or to slowor halt the progression of, the condition being treated (See, e.g.,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics,McGraw-Hill, New York, N.Y., the contents of which are incorporatedherein by reference, for a general description of the methods foradministering various agents for human therapy). The compositionscomprising the disclosed antibodies and agents can be delivered usingcontrolled or sustained-release delivery systems (e.g., capsules,biodegradable matrices). Examples of delayed-release delivery systemsfor drug delivery that would be suitable for administration of thecompositions of the disclosed compounds are described in, e.g., U.S.Pat. Nos. U.S. Pat. Nos. 5,990,092; 5,039,660; 4,452,775; and 3,854,480,the entire teachings of which are incorporated herein by reference.

For preparing pharmaceutical compositions from the anti-VISTA antibodiesand/or fragments of the present invention, pharmaceutically acceptablecarriers can be solid or liquid. Solid form preparations includepowders, tablets, pills, capsules, cachets, suppositories, anddispersible granules. For example, the compounds of the presentinvention can be in powder form for reconstitution at the time ofdelivery. A solid carrier can be one or more substances which can alsoact as diluents, flavoring agents, solubilizers, lubricants, suspendingagents, binders, preservatives, tablet disintegrating agents, or anencapsulating material. In powders, the carrier is a finely dividedsolid which is in a mixture with the finely divided active ingredient.

The powders and tablets preferably contain from about one to aboutseventy percent of the active ingredient. Suitable carriers aremagnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcaboxymethylcellulose, a low-melting wax, cocoa butter, and the like.Tablets, powders, cachets, lozenges, fast-melt strips, capsules andpills can be used as solid dosage forms containing the active ingredientsuitable for oral administration.

Liquid form preparations include solutions, suspensions, retentionenemas, and emulsions, for example, water or water propylene glycolsolutions. For parenteral injection, liquid preparations can beformulated in solution in aqueous polyethylene glycol solution.

The pharmaceutical composition can be in unit dosage form. In such form,the composition is subdivided into unit doses containing appropriatequantities of the active ingredient. The unit dosage form can be apackaged preparation, the package containing discrete quantities of unitdoses. The dosages can be varied depending upon the requirements of thepatient, the severity of the condition being treated, the compound andthe route of administration being employed. Determination of the properdosage for a particular situation is within the skill in the art.

Also, the pharmaceutical composition can contain, if desired, othercompatible agents, e.g., pharmaceutical, therapeutic or prophylacticagents. Therapeutic or prophylactic agents include, but are not limitedto, peptides, polypeptides, proteins, fusion proteins, nucleic acidmolecules, small molecules, mimetic agents, synthetic drugs, inorganicmolecules, and organic molecules. Examples of the classes of such agents(e.g., anti-cancer agents) include, but are not limited to, cytotoxins,angiogenesis inhibitors, immunomodulatory agents, immuno-oncologyagents, and agents used to provide relief from pain or to offset thedeleterious effects of one or more therapeutic agents (e.g.,bisphosphonate use to reduce the hypercalcemic effects ofglucocorticoids).

Angiogenesis inhibitors, agents and therapies that are suitable for usein the compositions and methods described herein include, but are notlimited to, angiostatin (plasminogen fragment); antiangiogenicantithrombin III; angiozyme. Bisphosphonates include, but are notlimited to, alendronate, clodronate, etidronate, ibandronate,pamidronate, risedronate, tiludronate, and zoledronate.

Immunomodulatory agents and therapies that are suitable for use in thecompositions and methods described herein include, but are not limitedto, anti-T cell receptor antibodies such as anti-CD3 antibodies (e.g.Nuvion (Protein Design Labs), OKT3 (Johnson & Johnson), or anti-CD20antibodies Rituxan (IDEC)), anti-CD52 antibodies (e.g. CAMPATH 1H(Ilex)), anti-CD11a antibodies (e.g. Xanelim (Genentech)); anti-cytokineor anti-cytokine receptor antibodies and antagonists such as anti-IL-2receptor antibodies (Zenapax (Protein Design Labs)), anti-IL-6 receptorantibodies (e.g. MRA (Chugai)), and anti-IL-12 antibodies(CNT01275(Janssen)), anti-TNFalpha antibodies (Remicade(Janssen)) or TNFreceptor antagonist (Enbrel (Immunex)), anti-IL-6 antibodies (BE8(Diaclone) and siltuximab (CNTO32 (Centocor)), and antibodies thatimmunospecifically bind to tumor-associated antigens (e.g., trastuzimab(Genentech)).

Immuno-oncology agents that are suitable for use in the compositions andmethods described herein include, but are not limited to, ipilimumab(anti-CTLA-4), nivolumab (anti-PD-1), pembrolizumab (anti-PD-1),anti-PD-L1 antibodies, and anti-LAG-3 antibodies.

The composition is preferably made in the form of a dosage unitcontaining a therapeutically effective amount of the antibody orfragment. Examples of dosage units are tablets and capsules. Fortherapeutic purposes, the tablets and capsules can contain, in additionto the active ingredient, conventional carriers such as binding agents,for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, ortragacanth; fillers, for example, calcium phosphate, glycine, lactose,maize-starch, sorbitol, or sucrose; lubricants, for example, magnesiumstearate, polyethylene glycol, silica, or talc; disintegrants, forexample potato starch, flavoring or coloring agents, or acceptablewetting agents. Oral liquid preparations generally in the form ofaqueous or oily solutions, suspensions, emulsions, syrups or elixirs cancontain conventional additives such as suspending agents, emulsifyingagents, non-aqueous agents, preservatives, coloring agents and flavoringagents. Examples of additives for liquid preparations include acacia,almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucosesyrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose,methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, orsorbic acid.

Other general details regarding methods of making and using thecompounds and compositions described herein are well-known in the art.See, e.g., U.S. Pat. No. 7,820,169, the contents of which areincorporated in their entirely.

Methods of Treatment

One of skill in the art, e.g., a clinician, can determine the suitabledosage and route of administration for a particular antibody, fragmentor composition for administration to an individual, considering theagents chosen, pharmaceutical formulation and route of administration,various patient factors and other considerations. Preferably, the dosagedoes not cause or produces minimal or no adverse side effects. Instandard multi-dosing regimens, a pharmacological agent may beadministered on a dosage schedule that is designed to maintain apre-determined or optimal plasma concentration in the subject undergoingtreatment. The antibodies, fragments and compositions can be added atany appropriate dosage ranges or therapeutically effective amount, forexample, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg,2.5 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0mg/kg, 9.0 mg/kg, 10.0 mg/kg, 11.0 mg/kg, 12.0 mg/kg, 13.0 mg/kg, 14.0mg/kg, 15.0 mg/kg, 16.0 mg/kg, 17.0 mg/kg, 18.0 mg/kg, 19.0 mg/kg, 20.0mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg 60 mg/kg, 70 mg/kg, 80 mg/kg, 90mg/kg and 100 mg/kg. In one embodiment, the dosage of the administeredcomposition, antibody or fragment is 0.1-15 mg/kg per administration.

The antibody or fragment can be administered once, at least once, twice,at least twice, three times, or at least three times per day. Theantibody or fragment can be administered once, at least once, twice, atleast twice, three times, at least three times, four times, at leastfour times, five times, at least five times, six times per week, or atleast six times per week. The antibody or fragment can be administeredonce per month, at least once per month, twice per month, at least twiceper month, three times per month or at least three times per month. Theantibody or antibody fragment can be administered once per year, atleast once per year, twice per year, at least twice per year, threetimes per year, at least three times per year, four times per year, atleast four times per year, five times per year, at least five times peryear, six times per year or at least six times per year.

The anti-VISTA antibodies, fragments and compositions can, for example,be administered through parenteral or nonparenteral means, including,but not limited to, intravenously, subcutaneously, orally, rectally,intramuscularly, intraperitoneally, transmucosally, transdermally,intrathecally, nasally, or topically. One of ordinary skill in the artwill recognize that the following dosage forms can comprise as theactive ingredient, either compounds or a corresponding pharmaceuticallyacceptable salt of a compound of the present invention. In someembodiments, the dosage forms can comprise as the active ingredient,either a compound or a corresponding pharmaceutically acceptable salt ofa compound.

The anti-VISTA antibodies of the invention can be administered as partof a combination therapy (e.g., with each other, or with one or moreother therapeutic agents). The compounds of the invention can beadministered before, after or concurrently with one or more othertherapeutic agents. In some embodiments, a compound of the invention andother therapeutic agent can be co-administered simultaneously (e.g.,concurrently) as either separate formulations or as a joint formulation.Alternatively, the agents can be administered sequentially, as separatecompositions, within an appropriate time frame, as determined by theskilled clinician (e.g., a time sufficient to allow an overlap of thepharmaceutical effects of the therapies). A compound of the inventionand one or more other therapeutic agents can be administered in a singledose or in multiple doses, in an order and on a schedule suitable toachieve a desired therapeutic effect.

The present invention also provides a method for modulating or treatingat least one malignant disease in a cell, tissue, organ, animal orpatient. In some embodiments, the compounds and compositions of thepresent invention are used to treat or prevent cancer. Cancer caninclude any malignant or benign tumor of any organ or body system.Examples include, but are not limited to, the following: breast,digestive/gastrointestinal, endocrine, neuroendocrine, eye,genitourinary, germ cell, gynecologic, head and neck, hematologic/blood,musculoskeletal, neurologic, respiratory/thoracic, bladder, colon,rectal, lung, endometrial, kidney, pancreatic, liver, stomach,testicular, esophageal, prostate, brain, cervical, ovarian and thyroidcancers. Other cancers can include leukemias, melanomas, and lymphomas,and any cancer described herein. In some embodiments, the solid tumor isinfiltrated with myeloid and/or T-cells. In some embodiments, the canceris a leukemia, lymphoma, myelodysplastic syndrome and/or myeloma. Insome embodiments, the cancer can be any kind or type of leukemia,including a lymphocytic leukemia or a myelogenous leukemia, such as,e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia(CLL), acute myeloid (myelogenous) leukemia (AML), chronic myelogenousleukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia,large granular lymphocytic leukemia, or adult T-cell leukemia. In someembodiments, the lymphoma is a histocytic lymphoma, follicular lymphomaor Hodgkin lymphoma, and in some embodiments, the cancer is a multiplemyeloma. In some embodiments, the cancer is a solid tumor, for example,a melanoma, or bladder cancer. In a particular embodiment, the cancer isa lung cancer, such as a non-small cell lung cancer (NSCLC).

The present invention also provides a method for modulating or treatingat least one malignant disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: leukemia, acuteleukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL,acute myeloid leukemia (AML), chronic myelocytic leukemia (CIVIL),chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplasticsyndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma,non-hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi'ssarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngealcarcinoma, malignant histiocytosis, paraneoplasticsyndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas,sarcomas, malignant melanoma, hemangioma, metastatic disease, cancerrelated bone resorption, cancer-related bone pain, and the like. In someembodiments, the solid tumor is infiltrated with myeloid and/or T-cells.In a particular embodiment, the solid tumor is a lung cancer, such as anon-small cell lung cancer (NSCLC).

In some embodiments, the compounds and therapies described herein areco-administered with a vaccine (such as a viral vector vaccine,bacterial vaccine, cell-based vaccine, DNA vaccine, RNA vaccine, peptidevaccine, or protein vaccine). Such vaccines are well known in the art.See, e.g., Jeffrey Schlom, “Therapeutic Cancer Vaccines: Current Statusand Moving Forward,” J Natl Cancer Inst; 104:599-613 (2012), thecontents of which are incorporated herein in their entirely.

In some embodiments, the compounds and therapies described herein areco-administered with agents for chemotherapy, hormone therapies andbiological therapies, and/or bisphosphonates. In some embodiments, theagent(s) for chemotherapy include one or more of the following:arboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ),cyclophosphamide (Cytoxan, Neosar), doxorubicin (Adriamycin), etoposide(VePesid), fluorouracil (5-FU), gemcitabine (Gemzar), irinotecan(Camptosar), paclitaxel (Taxol), topotecan (Hycamtin), vincristine(Oncovin, Vincasar PFS), vinblastine (Velban).

In other embodiments, the anti-VISTA compounds and therapies describedherein are co-administered with one or more immune checkpointantibodies, such as, for example, nivolumab, pembrolizumab,tremelimumab, ipilimumab, anti-PD-L1 antibody, anti-PD-L2 antibody,anti-TIM-3 antibody, anti-LAG-3, anti-OX40 antibody and anti-GITRantibody.

In another embodiment, the anti-VISTA compounds and therapies describedherein are co-administered with a small molecule inhibitor ofindoleamine 2,3-dioxygenase (IDO).

The anti-VISTA compounds and composition of the invention may beadministered to a subject in need thereof to prevent (includingpreventing the recurrence of cancer) or treat (e.g., manage orameliorate a cancer or one or more symptoms thereof) cancer. Any agentor therapy (e.g., chemotherapies, radiation therapies, targetedtherapies, such as imatinib, sorafenib and vemurafenib, hormonaltherapies, and/or biological therapies or immunotherapies) which isknown to be useful, or which has been used or is currently being usedfor the prevention, treatment, management or amelioration of cancer orone or more symptoms thereof can be used in combination with a compoundor composition of the invention described herein. Anti-cancer agents,but not limited to: 5-fluoruracil; acivicin; aldesleukin; altretamine;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;azacitidine; azetepa; azotomycin; batimastat; bicalutamide; bleomycinsulfate; brequinar sodium; bropirimine; busulfan; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicinhydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflornithine hydrochloride; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; fazarabine; fenretinide; floxuridine;fludarabine phosphate; fluorouracil; flurocitabine; fosquidone;fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea;idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II(including recombinant interleukin II, or rIL2), interferon alpha-2a;interferon alpha-2b; interferon alpha-m; interferon alpha-n3; interferonbeta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride;lanreotide acetate; letrozole; leuprolide acetate; liarozolehydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride;masoprocol; mechlorethamine hydrochloride; megestrol acetate;melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitomycin;mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid;nocodazole; ormaplatin; paclitaxel; pegaspargase; porfromycin;prednimustine; procarbazine hydrochloride; puromycin; rogletimide;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tegafur; teloxantrone hydrochloride; temoporfin;teniposide; teroxirone; testolactone; thiamiprine; thioguanine;thiotepa; tiazofurin; tirapazamine; topotecan; trimetrexate;trimetrexate glucuronate; triptorelin; uracil mustard; uredepa;vapreotide; verteporfn; vinblastine sulfate; vincristine sulfate;vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride. Targeted therapies include, but are not limited to,tyrosine kinase inhibitors (e.g., imatinib, sorafenib, and vemurafenib).The invention also encompasses administration of an anti-VISTA compoundof the invention in combination with radiation therapy comprising theuse of x-rays, gamma rays and other sources of radiation to destroy thecancer cells. Cancer treatments are known in the art and have beendescribed in such literature as the Physician's Desk Reference (57thed., 2003).

The anti-VISTA antibodies described herein are also useful, for example,in the treatment of chronic infectious diseases, such as HIV, HBV, HCV,and HSV, among others.

Various properties and sequence information for select anti-VISTAantibodies of the invention are provided in Tables 1A, 1B and 2 herein.

TABLE 1ACDR Sequences of Select Fully Human or Humanized anti-human VISTA antibodiesVH Heavy-chain Heavy-chain Heavy-chain Light-chain Light-chainLight-chain mAb ID family cdr1 (Imgt) cdr2 (Imgt) cdr3 (Imgt)cdr1 (Imgt) cdr2 (Imgt) cdr3 (Imgt) VSTB50 B GYTFTNYG INPYTGEPAREGYGNYIFPY ESVDTYANSL RAS QQTNEDPRT (SEQ ID (SEQ ID (SEQ ID NO: 3)(SEQ ID (SEQ ID (SEQ ID NO: 1) NO: 2) NO: 4) NO: 5) NO: 6) VSTB53GYTFTHYT IIPSSGYS ARGAYDDYYDYYAMDY QTIVHSNGNTY KVS FQASHVPWT (SEQ ID(SEQ ID (SEQ ID NO: 9) (SEQ ID (SEQ ID (SEQ ID NO: 7) NO: 8) NO: 10)NO: 11) NO: 12) VSTB60 B GYTFTNYG INTYTGES ARDYYGIYVSAY ESVDNYANSF RASQQSHEDPYT (SEQ ID (SEQ ID (SEQ ID NO: 15) (SEQ ID (SEQ ID (SEQ IDNO: 13) NO: 14) NO: 16) NO: 17) NO: 18) VSTB95 GFTFRNYG IISGGSYTARIYDHDGDYYAMDY QSIVHSNGNTY KVS FQGSHVPWT (SEQ ID (SEQ ID(SEQ ID NO: 21) (SEQ ID (SEQ ID (SEQ ID NO: 19) NO: 20) NO: 22) NO: 23)NO: 24) VSTB112 D GGTFSSYA IIPIFGTA ARSSYGWSYEFDY QSIDTR SAS QQSAYNPIT(SEQ ID (SEQ ID (SEQ ID NO: 27) (SEQ ID (SEQ ID (SEQ ID NO: 25) NO: 26)NO: 28) NO: 29) NO: 30) VSTB116 D GGTFSSYA IIPIFGTA ARSSYGWSYEFDY QSINTNAAS QQARDTPIT (SEQ ID (SEQ ID (SEQ ID NO: 33) (SEQ ID (SEQ ID (SEQ IDNO: 31) NO: 32) NO: 34) NO: 35) NO: 36)

TABLE 1B Heavy and Light Chain Sequences of Select Fully Human orHumanized anti-human VISTA antibodies  Protein ID Heavy-chain AA CDSLight-chain AA CDS VSTB50QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGLNWVRQAPGQGLEWDIVMTQTPLSLSVTPGQPASISCRASESVDMGWINPYTGEPTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYTYANSLMHWYLQKPGQPPQLLIYRASNLESYCAREGYGNYIFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVYCQQTNEDPRTFGQGTKLEIKRTVAAPSVFTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEIFPPSDEQLKSGTASVVCLLNNFYPREAKVLLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDQWKVDNALQSGNSQESVTEQDSKDSTYSLSGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKASTLTLSKADYEKHKVYACEVTHQGLSSPVTLPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP KSFNRGEC(SEQ ID NO: 48)SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 47) VSTB53QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYTIHWVRQAPGQGLEWDIVMTQSPLSLPVTPGEPASISCRSSQTIVMGYIIPSSGYSEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFYCARGAYDDYYDYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGVPDRFSGSGSGTDFTLKISRVEAEDVGVSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLYYCFQASHVPWTFGQGTKLEIKRTVAAPSVSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCFIFPPSDEQLKSGTASVVCLLNNFYPREAKPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVQWKVDNALQSGNSQESVTEQDSKDSTYSLWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSTLTLSKADYEKHKVYACEVTHQGLSSPVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK TKSFNRGEC(SEQ ID NO: 50)GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 49) VSTB60QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMTWVRQAPGQGLEWDIVMTQTPLSLSVTPGQPASISCRASESVDMGWINTYTGESTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYNYANSFMHWYLQKPGQSPQLLIYRASNLESYCARDYYGIYVSAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVYCQQSHEDPYTFGQGTKLEIKRTVAAPSVFTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEIFPPSDEQLKSGTASVVCLLNNFYPREAKVLLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDQWKVDNALQSGNSQESVTEQDSKDSTYSLSGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKASTLTLSKADYEKHKVYACEVTHQGLSSPVTLPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP KSFNRGEC(SEQ ID NO: 52)SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 51) VSTB95EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYGMSWVRQAPGKGLEWDIVMTQSPLSLPVTPGEPASISCRSSQSIVVASIISGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFYCARIYDHDGDYYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSSGVPDRFSGSGSGTDFTLKISRVEAEDVGVGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSYYCFQGSHVPVVTFGQGTKLEIKRTVAAPSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPVFIFPPSDEQLKSGTASVVCLLNNFYPREAAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWKVQWKVDNALQSGNSQESVTEQDSKDSTYSYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSLSSTLTLSKADYEKHKVYACEVTHQGLSSPNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGVTKSFNRGEC(SEQ ID NO: 54)FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 53) VSTB112QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWDIQMTQSPSSLSASVGDRVTITCRASQSIDMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYTRLNWYQQKPGKAPKLLIYSASSLQSGVPSYCARSSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGRFSGSGSGTDFTLTISSLQPEDFATYYCQQTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVSAYNPITFGQGTKVEIKRTVAAPSVFIFPPVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPSDEQLKSGTASVVCLLNNFYPREAKVQWKVELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDNALQSGNSQESVTEQDSKDSTYSLSSTLTDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKLSKADYEKHKVYACEVTHQGLSSPVTKSFNALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY RGEC(SEQ ID NO: 56)PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 55) VSTB116QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWDIQMTQSPSSLSASVGDRVTITCRASQSINMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYTNLNWYQQKPGKAPKLLIYAASSLQSGVPSYCARSSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGRFSGSGSGTDFTLTISSLQPEDFATYYCQQTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVARDTPITFGQGTKVEIKRTVAAPSVFIFPPVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPSDEQLKSGTASVVCLLNNFYPREAKVQWKVELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDNALQSGNSQESVTEQDSKDSTYSLSSTLTDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKLSKADYEKHKVYACEVTHQGLSSPVTKSFNALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY RGEC(SEQ ID NO: 58)PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 57) VSTB140*QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWDIQMTQSPSSLSASVGDRVTITCRASQSIDMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYTRLNWYQQKPGKAPKLLIYSASSLQSGVPSYCARSSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESRFSGSGSGTDFTLTISSLQPEDFATYYCQQTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVSAYNPITFGQGTKVEIKRTVAAPSVFIFPPVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAASDEQLKSGTASVVCLLNNFYPREAKVQWKVASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEDNALQSGNSQESVTEQDSKDSTYSLSSTLTVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSLSKADYEKHKVYACEVTHQGLSSPVTKSFNSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI RGEC(SEQ ID NO: 56)AVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 59) VSTB149*^(Δ)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWDIQMTQSPSSLSASVGDRVTITCRASQSIDMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYTRLNWYQQKPGKAPKLLIYSASSLQSGVPSYCARSSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGRFSGSGSGTDFTLTISSLQPEDFATYYCQQTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVSAYNPITFGQGTKVEIKRTVAAPSVFIFPPVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPSDEQLKSGTASVVCLLNNFYPREAKVQWKVPVAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDDNALQSGNSQESVTEQDSKDSTYSLSSTLTGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNAALSKADYEKHKVYACEVTHQGLSSPVTKSFNLPAPIAKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP RGEC(SEQ ID NO: 56)SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 60) VSTB174*QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWDIQMTQSPSSLSASVGDRVTITCRASQSIDMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYTRLNWYQQKPGKAPKLLIYSASSLQSGVPSYCARSSYGWSYEFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGRFSGSGSGTDFTLTISSLQPEDFATYYCQQTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVSAYNPITFGQGTKVEIKRTVAAPSVFIFPPVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPSDEQLKSGTASVVCLLNNFYPREAKVQWKVELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDNALQSGNSQESVTEQDSKDSTYSLSSTLTDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKLSKADYEKHKVYACEVTHQGLSSPVTKSFNALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY RGEC(SEQ ID NO: 56)PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 61) *Constant region sequences inVSTB140, VSTB149 and VSTB174 are underlined. ^(Δ)Amino acid residuesconferring protease resistance in the heavy chain of VSTB149 areindicated in bold.

TABLE 2 Dissociation constant (K_(D)) for select anti-VISTA antibodiesSample KD (M) ka1 (1/Ms) kd1 (1/s) S1 1.71E−10 1.69E+06 2.89E−041.09E−10 1.11E+06 1.21E−04 S40 5.07E−10 1.46E+05 7.40E−05 6.96E−101.39E+05 9.69E−05 S41 6.32E−10 4.82E+05 3.05E−04 3.10E−10 7.08E+052.19E−04 S42 1.04E−10 1.05E+06 1.09E−04 2.65E−10 5.13E+05 1.36E−04 S432.64E−11 1.25E+06 3.30E−05 5.28E−11 1.18E+06 6.22E−05 S44 2.53E−111.23E+06 3.12E−05 6.40E−11 9.93E+05 6.36E−05 S45 2.35E−11 1.58E+063.72E−05 2.58E−11 1.46E+06 3.77E−05 S46 1.06E−10 1.56E+06 1.66E−042.96E−10 1.50E+06 4.44E−04 S47 3.56E−10 5.14E+05 1.83E−04 2.52E−105.69E+05 1.43E−04 S33 8.30E−10 1.23E+06 1.02E−03 1.22E−09 8.96E+051.10E−03 S34 1.08E−09 5.95E+05 6.43E−04 2.80E−09 5.20E+05 1.46E−03 S358.06E−11 2.08E+06 1.68E−04 1.35E−10 1.78E+06 2.41E−04 S36 6.29E−111.77E+06 1.12E−04 2.90E−11 1.58E+06 4.58E−05 S37 2.23E−09 5.10E+051.14E−03 4.43E−09 3.94E+05 1.75E−03 S38 2.26E−09 5.18E+05 1.17E−032.03E−09 5.37E+05 1.09E−03 S39 5.62E−10 3.97E+05 2.23E−04 3.47E−104.15E+05 1.44E−04 S25 1.31E−09 6.21E+05 8.12E−04 1.10E−09 5.65E+056.24E−04 S26 No Binding 3.53E−09 2.38E+05 8.41E−04 S27 1.13E−09 8.86E+059.97E−04 1.61E−09 7.12E+05 1.15E−03 S48 3.12E−10 1.24E+06 3.87E−041.21E−09 8.78E+05 1.06E−03 S28 2.03E−09 1.08E+06 2.19E−03 2.03E−099.30E+05 1.88E−03 S29 3.78E−11 1.42E+06 5.38E−05 8.90E−11 9.06E+058.06E−05 S30 No Binding No Binding S31 Weak Binding Weak Binding S32Weak Binding Weak Binding S15 9.34E−11 6.46E+05 6.04E−05 5.13E−103.50E+05 1.80E−04 S16 1.26E−10 5.54E+05 6.99E−05 1.92E−10 4.43E+058.53E−05 S17 7.68E−10 9.88E+05 7.59E−04 4.10E−10 7.09E+05 2.91E−04 S182.28E−09 4.90E+05 1.12E−03 1.05E−09 3.13E+05 3.29E−04 S19 1.54E−091.02E+06 1.58E−03 2.86E−10 7.03E+05 2.01E−04 S20 1.48E−09 6.67E+059.85E−04 4.57E−10 6.36E+05 2.91E−04 S21 3.18E−09 3.16E+05 1.00E−031.34E−09 2.70E+05 3.60E−04 S22 2.98E−09 1.09E+06 3.25E−03 1.27E−091.25E+06 1.59E−03 S6 6.36E−10 5.28E+05 3.36E−04 3.02E−10 5.98E+051.80E−04 S7 6.75E−10 1.31E+06 8.87E−04 3.27E−10 1.15E+06 3.75E−04 S81.15E−10 1.89E+06 2.18E−04 5.97E−11 1.25E+06 7.48E−05 S9 1.67E−101.87E+06 3.11E−04 9.31E−11 1.27E+06 1.18E−04 S10 8.90E−11 1.55E+061.38E−04 4.30E−11 1.22E+06 5.27E−05 S12 4.94E−10 1.57E+06 7.76E−042.39E−10 1.19E+06 2.86E−04 S13 1.02E−10 1.42E+06 1.44E−04 6.46E−119.55E+05 6.17E−05 S14 2.02E−10 1.26E+06 2.55E−04 7.55E−11 1.12E+068.43E−05 S1 2.06E−10 1.60E+06 3.29E−04 8.35E−11 1.21E+06 1.01E−04 S21.56E−10 9.74E+05 1.52E−04 8.66E−11 7.25E+05 6.28E−05 S3 4.33E−119.07E+05 3.93E−05 4.89E−11 7.41E+05 3.63E−05 S4 1.52E−10 8.98E+051.36E−04 7.54E−11 6.93E+05 5.23E−05 S49 1.45E−10 1.01E+06 1.46E−041.04E−10 7.28E+05 7.60E−05 S5 2.13E−10 1.25E+06 2.67E−04 1.37E−108.51E+05 1.17E−04

Methods of Eliciting a Biological Response

In an embodiment, the present invention provides a method of eliciting abiological response in a subject using an antibody that binds a V-domainIg Suppressor of T cell Activation (VISTA) protein. The method comprisesthe steps of administering to a subject an antibody that binds a VISTAprotein, or an antigen-binding fragment thereof, in an amount sufficientto elicit a biological response in the subject.

In certain embodiments, the biological response that is elicited by theantibody, or antigen-binding fragment, that binds VISTA is a decrease inthe number of circulating immune cells; a decrease in the number ofgranulocytes in bone marrow and/or spleen; an increase in the number ofneutrophils, macrophages, T cells, or a combination thereof in a tumormicroenvironment (TME); an increase in the level of one or morecytokines; or any combination of these responses.

In an embodiment, the biological response that is elicited is a decreasein the number of circulating immune cells. The circulating immune cellsthat are decreased can be, for example, monocytes, neutrophils,lymphoctyes, eosinophils, basophils, or any combination thereof. In oneembodiment, the decrease in the number of circulating immune cells istransient.

As used herein, a “transient” decrease in the number of circulatingimmune cells refers to a temporary decrease relative to a level prior toadministration of the antibody, or antigen-binding fragment, wherein thedecreased level is restored to, or surpasses, the prior level at asubsequent time point.

In an embodiment, the biological response that is elicited is a decreasein the number of granulocytes in one or more tissues (e.g., bone marrow)and/or organs (e.g., spleen) of the subject.

In an embodiment, the biological response that is elicited is anincrease in the number of immune cells in a tumor microenvironment(TME). The immune cells that are increased in the TME can include, butare not limited to, neutrophils, macrophages, T cells, or anycombination thereof.

In an embodiment, the biological response that is elicited is anincrease in the level of one or more cytokines (e.g., one or morechemokines). Examples of cytokines that can be increased in response toadministration of an anti-VISTA antibody or antigen-binding fragmentinclude, for example, IL-6, TNFα, MCP-3, MDC, MIP-1β, IP-10, IL-1Rα,GM-CSF, IL-12p70, GRO, MIP-1α, IL-1β, RANTES, G-CSF, IL-1α, IL-7,IL-12p40, IL-13, IFNγ, TNFβ, IFNα, IL-4, IL-10, FGF-2, fractalkine,VEGF, IL-17A, Flt3L, IL-9, TGFa, IL-15, EGF, PDGF-αα, MCP-1, IL-8,sCD40L, eotaxin, IL-2, IL-3, and IL-5, and PDGF-BB.

In a particular embodiment, the antibody that binds VISTA, orantigen-binding fragment thereof, that is administered to the subjectcomprises an Fc region having effector function (e.g., binds to an Fcreceptor on a cell). In a particular embodiment, the antibody orantigen-binding fragment thereof, binds to a CD16 receptor (e.g.,FcγRIIIa, FcγRIIIb) on an immune cell (e.g., NK cell).

In an embodiment, the antibody that binds VISTA, or antigen-bindingfragment thereof, that is administered to the subject comprises anantibody VH domain comprising a VH CDR1 having the amino acid sequenceof SEQ ID NO:25, a VH CDR2 having the amino acid sequence of SEQ IDNO:26 and a VH CDR3 having the amino acid sequence of SEQ ID NO:27, andwhich further comprises an antibody VL domain comprising a VL CDR1having the amino acid sequence of SEQ ID NO:28, a VL CDR2 having theamino acid sequence of SEQ ID NO:29 and a VL CDR3 having the amino acidsequence of SEQ ID NO:30. In a particular embodiment, the antibody isVSTB174, or an antigen-binding fragment thereof.

In an embodiment, the subject to which the antibody that binds VISTA, orantigen-binding fragment thereof, is administered is a mammal. In oneembodiment, the mammal is a human. In another embodiment, the mammal isa non-human primate. In yet another embodiment, the mammal is a rodent(e.g., a mouse, a rat).

In an embodiment, the subject has cancer (e.g., a solid tumor, aleukemia, a lymphoma). In some embodiments, the cancer is a leukemia,lymphoma, myelodysplastic syndrome and/or myeloma. In some embodiments,the cancer can be any kind or type of leukemia, including a lymphocyticleukemia or a myelogenous leukemia, such as, e.g., acute lymphoblasticleukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid(myelogenous) leukemia (AML), chronic myelogenous leukemia (CIVIL),hairy cell leukemia, T-cell prolymphocytic leukemia, large granularlymphocytic leukemia, or adult T-cell leukemia. In some embodiments, thelymphoma is a histocytic lymphoma, and in some embodiments, the canceris a multiple myeloma. In some embodiments, the cancer is a solid tumor,for example, a melanoma, a breast cancer or bladder cancer. In someembodiments, the cancer is a lung cancer (e.g., a non-small cell lungcarcinoma (NSCLC)). In some embodiments, the cancer is a bladder cancer.In some embodiments, the cancer is a breast cancer.

The antibody or fragment thereof can be administered by any suitableparenteral or nonparenteral means including, for example, intravenously(IV), subcutaneously (SQ) or orally (PO).

Methods of Screening for Anti-Vista Antibodies

The present invention provides, in an embodiment, a method ofidentifying an antibody that binds a V-domain Ig Suppressor of T cellActivation (VISTA) protein and elicits a biological response. The methodcomprises the steps of providing an antibody that binds VISTA, or anantibody fragment thereof, to e.g., a cell, tissue, organ and/ororganism, and determining whether the antibody or antibody fragmentthereof induces a biological response in the cell, tissue, organ ororganism.

In an embodiment, the biological response to be determined includesactivation of monocytes; activation of T cells; a decrease in the numberof circulating immune cells; a decrease in the number of granulocytes inbone marrow and spleen; an increase in the number of neutrophils,macrophages, T cells, or a combination thereof in a tumormicroenvironment (TME); an increase in the level of one or morecytokines; or any combination of these responses.

In an embodiment, the antibody that binds VISTA, or antigen-bindingfragment thereof, to be identified according to the present methodcomprises an Fc region having effector function (e.g., binds to an Fcreceptor on a cell). In a particular embodiment, the antibody orantigen-binding fragment thereof, binds to a CD16 receptor (e.g.,FcγRIIIa, FcγRIIIb) on an immune cell (e.g., NK cell).

In an embodiment, the antibody that binds VISTA, or antigen-bindingfragment thereof, to be identified according to the present methodcomprises an antibody VH domain comprising a VH CDR1 having the aminoacid sequence of SEQ ID NO:25, a VH CDR2 having the amino acid sequenceof SEQ ID NO:26 and a VH CDR3 having the amino acid sequence of SEQ IDNO:27, and which further comprises an antibody VL domain comprising a VLCDR1 having the amino acid sequence of SEQ ID NO:28, a VL CDR2 havingthe amino acid sequence of SEQ ID NO:29 and a VL CDR3 having the aminoacid sequence of SEQ ID NO:30.

In an embodiment, the biological response can be assayed in varioustypes of samples, including but not limited to, a tissue sample, abiological fluid sample (e.g. mammalian plasma, serum, lymph, wholeblood, spinal, amniotic, or other animal-derived fluid), a cell(s)(e.g., a tumor cell, an immune cell) sample, and the like. Samples caninclude, for instance: (a) preparations comprising un-fixed freshtissues and/or cells; (b) fixed and embedded tissue specimens, such asarchived material; and (c) frozen tissues or cells. Thus, samples can befresh or preserved, for example, in liquid solution, flash-frozen orlyophilized, smeared or dried, embedded, or fixed on slides or othersupports.

In some embodiments, tissue or cell samples are fixed or embedded.Fixatives are used, for example, to preserve cells and tissues in areproducible and life-like manner. Fixatives also stabilize cells andtissues, thereby protecting them from the rigors of processing andstaining techniques. For example, samples comprising tissue blocks,sections, or smears can be immersed in a fixative fluid, or in the caseof smears, dried.

Any means of sampling from a subject, for example, by blood draw, spinaltap, tissue smear or scrape, or tissue biopsy can be used to obtain asample. Thus, the sample can be a biopsy specimen (e.g., tumor, polyp,mass (solid, cell)), aspirate, smear or blood sample. The sample can bea tissue that has a tumor (e.g., cancerous growth) and/or tumor cells,or is suspected of having a tumor and/or tumor cells. For example, atumor biopsy can be obtained in an open biopsy, a procedure in which anentire (excisional biopsy) or partial (incisional biopsy) mass isremoved from a target area. Alternatively, a tumor sample can beobtained through a percutaneous biopsy, a procedure performed with aneedle-like instrument through a small incision or puncture (with orwithout the aid of a imaging device) to obtain individual cells orclusters of cells (e.g., a fine needle aspiration (FNA)) or a core orfragment of tissues (core biopsy).

The samples can be examined cytologically (e.g., smear), histologically(e.g., frozen or paraffin section) or using any other suitable method(e.g., molecular diagnostic methods). A tumor sample can also beobtained by in vitro harvest of cultured human cells derived from anindividual's tissue. Tumor samples can, if desired, be stored beforeanalysis by suitable storage means that preserve a sample's proteinand/or nucleic acid in an analyzable condition, such as quick freezing,or a controlled freezing regime. If desired, freezing can be performedin the presence of a cryoprotectant, for example, dimethyl sulfoxide(DMSO), glycerol, or propanediol-sucrose. Tumor samples can be pooled,as appropriate, before or after storage for purposes of analysis.

In an embodiment, the biological response can be determined using an invitro assay including, for example, immunological and immunochemicalmethods including, but not limited to, flow cytometry (e.g., FACSanalysis), a cytokine release assay, a chemokine release assay, a cellactivation assay, a cell proliferation assay, a cell migration assay,enzyme-linked immunosorbent assays (ELISA), including chemiluminescenceassays, radioimmunoassay, immunoblot (e.g., Western blot),immunohistochemistry (IHC), immunoprecipitation and other antibody-basedquantitative methods (e.g., Luminex® beads-based assays). Other suitablemethods include, for example, mass spectroscopy.

In an embodiment, the biological response can be determined in vivo in anon-human animal. In one embodiment, the non-human animal is a non-humanprimate. In yet another embodiment, the non-human animal is a rodent(e.g., a mouse, a rat). In some embodiments, the non-human animal is atransgenic animal.

In an embodiment, the non-human animal has cancer (e.g., a solid tumor,a leukemia, a lymphoma). In some embodiments, the cancer is a leukemia,lymphoma, myelodysplastic syndrome and/or myeloma. In some embodiments,the cancer can be any kind or type of leukemia, including a lymphocyticleukemia or a myelogenous leukemia, such as, e.g., acute lymphoblasticleukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid(myelogenous) leukemia (AML), chronic myelogenous leukemia (CIVIL),hairy cell leukemia, T-cell prolymphocytic leukemia, large granularlymphocytic leukemia, or adult T-cell leukemia. In some embodiments, thelymphoma is a histocytic lymphoma, and in some embodiments, the canceris a multiple myeloma. In some embodiments, the cancer is a solid tumor,for example, a melanoma, a breast cancer or bladder cancer. In someembodiments, the cancer is a lung cancer (e.g., a non-small cell lungcarcinoma (NSCLC)). In some embodiments, the cancer is a bladder cancer.In some embodiments, the cancer is a breast cancer.

In an embodiment, the biological response to be assayed is a decrease inthe number of circulating immune cells. The circulating immune cellsthat are decreased can be, for example, monocytes, neutrophils,lymphoctyes, eosinophils, basophils, or any combination thereof. In oneembodiment, the decrease in the number of circulating immune cells istransient.

In an embodiment, the biological response to be assayed is a decrease inthe number of granulocytes in one or more tissues (e.g., bone marrow) ororgans (e.g., spleen) of the subject.

In an embodiment, the biological response to be assayed is an increasein the number of immune cells in a tumor microenvironment (TME). Theimmune cells that are increased in the TME can include, but are notlimited to, neutrophils, macrophages, T cells, or any combinationthereof.

In an embodiment, the biological response to be assayed is an increasein the level of one or more cytokines (e.g., one or more chemokines).Examples of cytokines that can be increased in response toadministration of an anti-VISTA antibody or antigen-binding fragmentinclude, for example, IL-6, TNFα, MCP-3, MDC, MIP-1β, IP-10, IL-1Rα,GM-CSF, IL-12p70, GRO, MIP-1α, IL-1β, RANTES, G-CSF, IL-1α, IL-7,IL-12p40, IL-13, IFNγ, TNFβ, IFNα, IL-4, IL-10, FGF-2, fractalkine,VEGF, IL-17A, Flt3L, IL-9, TGFα, IL-15, EGF, PDGF-αα, MCP-1, IL-8,sCD40L, eotaxin, IL-2, IL-3, and IL-5, and PDGF-BB.

Anti-Vista Antibody Combination Therapies and Compositions

As described herein, certain immune responses can be enhanced using acombination of an anti-VISTA antibody of the invention with one or moreagents (e.g., antibodies) that bind to one or more immune checkpointproteins. Accordingly, in an embodiment, the present invention providesa pharmaceutical composition comprising a) an antibody or antibodyfragment thereof comprising an antigen binding region that binds toVISTA; b) an antibody or antibody fragment thereof comprising an antigenbinding region that binds to an immune checkpoint protein; and c) apharmaceutically acceptable carrier, diluent, or excipient.

In some embodiments, the antibody or antibody fragment thereofcomprising an antigen binding region that binds to an immune checkpointprotein maintains or enhances T cell activation in an individual. Asreadily understood by those of skill in the art, T cell activation caninclude, for example, an increase in the number of T cells and/or anincrease in T cell function. Methods of determining an increase in Tcell number, or an increase in T cell function (e.g., as determined by adecrease or increase in one or more markers of immune activation) can bereadily determined by those of skill in the art, and as describedherein.

Immune checkpoint proteins are known in the art, and include, forexample, PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, OX40 and GITR.Examples of antibodies that are known to bind to an immune checkpointprotein include, e.g., nivolumab, pembrolizumab, tremelimumab, andipilimumab.

In an embodiment, the pharmaceutical composition comprises a bispecificantibody that comprises the antibody or antibody fragment thereof thatbinds to VISTA and the antibody or antibody fragment thereof that bindsto an immune checkpoint protein. For example, the bispecific antibodycan comprise an anti-VISTA antibody of the present invention, and anantibody or antibody fragment thereof comprising an antigen bindingregion that binds to an immune checkpoint protein selected from, e.g.,PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, OX40 and GITR.

In one embodiment, the antibody or antibody fragment thereof that bindsto VISTA comprises an antibody VH domain comprising a VH CDR1 having theamino acid sequence of SEQ ID NO:25, a VH CDR2 having the amino acidsequence of SEQ ID NO:26 and a VH CDR3 having the amino acid sequence ofSEQ ID NO:27, and which further comprises an antibody VL domaincomprising a VL CDR1 having the amino acid sequence of SEQ ID NO:28, aVL CDR2 having the amino acid sequence of SEQ ID NO:29 and a VL CDR3having the amino acid sequence of SEQ ID NO:30. In some embodiments, theantibody or antibody fragment thereof that binds to VISTA comprises anantibody VH domain comprising SEQ ID NO:37. In some embodiments, theantibody or antibody fragment thereof that binds to VISTA comprises anantibody VL domain comprising SEQ ID NO:44.

In some embodiments, the antibody or antibody fragment thereof thatbinds to VISTA comprises an antibody heavy chain comprising SEQ ID NO:61and an antibody light chain comprising SEQ ID NO:56.

In an embodiment, the present invention provides a pharmaceuticalcomposition comprising a) an antibody or antibody fragment thereofcomprising an antigen binding region that binds to VISTA; b) an antibodyor antibody fragment thereof comprising an antigen binding region thatbinds to a PD-1 protein; and c) a pharmaceutically acceptable carrier,diluent, or excipient.

In some embodiments, the antibody or antibody fragment thereof thatbinds to VISTA comprises an antibody VH domain comprising a VH CDR1having the amino acid sequence of SEQ ID NO:25, a VH CDR2 having theamino acid sequence of SEQ ID NO:26 and a VH CDR3 having the amino acidsequence of SEQ ID NO:27, and which further comprises an antibody VLdomain comprising a VL CDR1 having the amino acid sequence of SEQ IDNO:28, a VL CDR2 having the amino acid sequence of SEQ ID NO:29 and a VLCDR3 having the amino acid sequence of SEQ ID NO:30.

In an embodiment, the antibody or antibody fragment thereof that bindsto VISTA comprises an antibody heavy chain comprising SEQ ID NO:61 andan antibody light chain comprising SEQ ID NO:56.

Various antibodies that bind to PD-1 are known in the art and include,for example, nivolumab and pembrolizumab.

In an embodiment, the present invention also provides a method ofenhancing an immune response in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a) an antibody or an antibody fragment thereof comprising anantigen binding region that binds VISTA; and b) an antibody or antibodyfragment thereof comprising an antigen binding region that binds to animmune checkpoint protein, thereby enhancing an immune response to thecancer.

In some embodiments, the antibody or antibody fragment thereofcomprising an antigen binding region that binds to an immune checkpointprotein maintains or enhances T cell activation.

In an embodiment, the antibody or antibody fragment thereof that bindsto VISTA comprises an antibody VH domain comprising a VH CDR1 having theamino acid sequence of SEQ ID NO:25, a VH CDR2 having the amino acidsequence of SEQ ID NO:26 and a VH CDR3 having the amino acid sequence ofSEQ ID NO:27, and which further comprises an antibody VL domaincomprising a VL CDR1 having the amino acid sequence of SEQ ID NO:28, aVL CDR2 having the amino acid sequence of SEQ ID NO:29 and a VL CDR3having the amino acid sequence of SEQ ID NO:30. In some embodiments, theantibody or antibody fragment thereof that binds to VISTA comprises anantibody VH domain comprising SEQ ID NO:37. In an embodiment, theantibody or antibody fragment thereof that binds to VISTA comprises anantibody VL domain comprising SEQ ID NO:44. In certain embodiments, theantibody or antibody fragment thereof that binds to VISTA comprises anantibody heavy chain comprising SEQ ID NO:61 and an antibody light chaincomprising SEQ ID NO:56.

In some embodiments, the antibody or antibody fragment thereof thatbinds to an immune checkpoint protein binds to an immune checkpointprotein selected from, e.g., PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3,OX40 and GITR. In one embodiment, the antibody that binds to an immunecheckpoint protein is, e.g., nivolumab, pembrolizumab, tremelimumab, oripilimumab.

In some embodiments, the immune response is an antitumor immuneresponse. In certain embodiments, the immune response is a T cellresponse.

In one embodiment, the individual has a cancer, such as, for example, asolid tumor (e.g., lung tumor, bladder tumor or breast tumor), aleukemia, a lymphoma, a myelodisplastic syndrome or a myeloma.

An anti-VISTA antibody of the present invention can be administeredbefore, after or concurrently with one or more antibodies (e.g., ananti-PD-1 antibody), or fragment thereof, that bind to an immunecheckpoint protein. In some embodiments, the anti-VISTA antibody of thepresent invention and one or more antibodies that bind to an immunecheckpoint protein can be co-administered simultaneously (e.g.,concurrently), as either separate formulations or as a jointformulation. Alternatively, the anti-VISTA antibody of the presentinvention and one or more antibodies that bind to an immune checkpointprotein can be administered sequentially, as separate formulations,within an appropriate time frame, as determined by the skilled clinician(e.g., a time sufficient to allow an overlap of the pharmaceuticaleffects of the therapies). An anti-VISTA antibody of the invention andone or more antibodies that bind an immune checkpoint protein can beadministered in a single dose or in multiple doses, in an order and on aschedule suitable to achieve a desired therapeutic effect.

EXAMPLES Example 1: Analysis of Vista Expression on Human HematopoieticCells

Methods:

Preparation and Staining of Fresh Human PBMCs For VISTA Expression

Expression of VISTA was tested on freshly isolated human PBMCs(peripheral blood mononuclear cells) from several donors. Anti-HumanVISTA-biotin (GA-1) was used for staining (5 μg/ml). Mouse IgG1,K-biotin (Clone MOPC-21 at 5 μg/ml) was used as an isotype control.

Donor Material

Blood samples were obtained from Biological Specialty Corp. (Colmar,Pa.) and were collected and analyzed the same day. 10 ml of whole bloodcontaining heparin sulfate were couriered for analysis.

Sample Preparation

Blood was diluted 1:1 in sterile PBS. 22 ml diluted cord blood waslayered onto 20 ml sterile Ficoll-Hypaque (GE Healthcare Cat #17-144003)in 50 ml conical tubes. Tubes were centrifuged at 1800 rpm for 20minutes at room temperature. Mononuclear cells at the interfacefollowing centrifugation were harvested using a 1 ml pipettor andcombined into two 50 ml conical tubes. Sterile PBS was added to eachtube to make the volume up to 50 ml and the cells were centrifuged at300 g for 10 minutes at 4° C. Supernatant was discarded. Cells wereresuspended in 50 ml of sterile PBS and tubes were spun at 300 g for 10minutes at 4° C. Supernatant was discarded. Cells were combined andresuspended in 50 ml sterile PBS prior to counting.

Staining Protocol: A frozen vial containing 5×10⁷ PBMCs was used forcompensation controls and as a control for staining.

The following reagents and/or consumables were used:

FACS Stain Buffer (BSA) from BD Biosciences (Cat #554657) supplementedwith 0.2% EDTA; Phosphate-Buffered saline (PBS) (Gibco cat #14190);96-well polypropylene round-bottomed plate (BD #3077); 1.2 mlpolypropylene cluster tubes (Corning #4451); biotinylated Anti-VISTAclone GA-1 from ImmunoNext Lot #080612B (used at 5 μg/ml); biotinylatedmIgG1, K isotype control (Clone MOPC-21); Biolegend cat #400104, Lot#B116649 (used at 5 μg/ml); anti-human antibodies (See staining tablebelow); near-Infrared live/dead dye (Invitrogen, cat #L10119); andstreptavidin reagents including STP-APC (BD Biosciences cat #554067, Lot#04251) (used at 1:200 dilution in FACS buffer), STP-PE (Biolegend cat#405203, Lot #B139688) (used at 1:200 dilution in FACS buffer), STP-PECy7 (showed non-specific binding in isotype control samples), STP-Q605(Invitrogen cat #Q10101MP, Lot #53449A) (used at 1:200 dilution in FACSbuffer).

Cell Surface Staining Protocol

Prior to staining, 1×10⁶ cells were transferred into 96-wellround-bottomed plates and were washed with 150 μl PBS. Plates were thencentrifuged at 1300 rpm at 4° C. for 3 minutes.

Subsequently, cells were washed again in PBS and centrifuged asdescribed above.

Live/dead staining was then performed in 50 μl PBS containing 0.25 μl ofnear-infrared live/dead dye. After 10 minutes at room temperature thewells were washed with 150 μl FACs staining buffer and centrifuged at1300 rpm at 4° C. for 3 minutes. Supernatant was discarded.

Cells were blocked with human serum at 1:100 in 50 μl FACS stainingbuffer. Plates were incubated at 4° C. for 15 minutes. Wells were thenwashed with 150 μl FACs staining buffer and centrifuged at 1300 rpm at4° C. for 3 minutes. Supernatant was discarded.

A cocktail containing the following antibodies was then added to eachwell for surface staining: The cocktails are described in Tables 3-6below. Each cocktail would be utilized separately from the othersdepending on the populations of interest.

TABLE 3 Lineage Stain Titer Target (μl/10⁶ Fluoro Antigen Mouse RatHuman Isotype Clone Supplier Cat No. Lot No. Cells) FITC/ CD19 X mlgG1HIB19 Biolegend 302206 B123019 2 AF488 PE CD11b X mlgG1, K ICRF44 BDBio. 555388 45134 2 PerCP- HLA-DR X mlgG2a, G46-6 BD Bio. 560652 251610.5 Cy5.5 K PE Cy7 CD16 X mlgG1, K 3G8 BD Bio. 557744 87825 0.2 APC Cy7NIR X Live/Dead AF700 CD56 X mlgG1, K B159 BD Bio. 557919 19470 1APC/AF647 VISTA-Bio X PB/V450 CD3 X mlgG1, K UCHT1 BD Bio. 558117 909260.5 Q605 CD14 X mlgG2a, TuK4 Invitrogen Q10013 1049158 0.2 K

TABLE 4 T Cell Stain Titer Target (μl/10⁶ Fluoro Antigen Mouse Rat HumanIsotype Clone Supplier Cat No. Lot No. Cells) FITC/AF488 CD4 X mlgG1,RPA-T4 BD Bio. 555346 38460 2 K PE VISTA- X Bio PerCP- CD8 X mlgG1,RPA-T8 BD Bio. 560662 1037 0.5 Cy5.5 K PE Cy7 CD56 X mlgG1, B159 BD Bio.557747 47968 0.5 K APC Cy7 NIR X AF700 CD45RO X mlgG2a, UCHL1 Biolegend304218 B143062 1 K APC/AF647 TCRgd X mlgG, B1 Biolegend 331212 B126473 2K PB/V450 CD45RA X mlgG2b, HI100 BD Bio. 560363 90928 0.5 K Q655 CD3 XmlgG2a 54.1 Invitrogen Q10012 982352 0.5

TABLE 5 DC Stain Titer Target (μl/10⁶ Fluoro Antigen Mouse Rat HumanIsotype Clone Supplier Cat No. Lot No. Cells) FITC/AF488 Lin1 X Mix MixBD Bio. 340546 2152758 5 PE CD11c X mlgG1, BD Bio. 555392 45123 2 KPerCP- HLA-DR X mlgG2, G46-6 BD Bio. 560662 25161 0.5 Cy5.5 K APC Cy7NIR X APC/AF647 CD83 X mlgG1a, HB15e BD Bio. 551073 57688 2 K BV421CD123 X mlgG1, 6H6 Biolegend 306017 B148193 0.5 K Q605 VISTA-Bio X

TABLE 6 Myeloid Titer Target (μl/10⁶ Fluoro Antigen Mouse Rat HumanIsotype Clone Supplier Cat No. Lot No. Cells) FITC/AF488 CD33 X mlgG1HM3-4 Biolegend 303304 B100963 3 PE CD11b X mlgG1, ICRF44 BD Bio. 55538845134 2 K APC Cy7 NIR X APC/AF647 VISTA-Bio X Q605 CD45 X mlgG1, HI30Invitrogen Q10051 880470 1 K

Following the surface staining, cells were washed twice as previouslydescribed with FACS staining buffer and centrifuged at 1300 rpm at 4° C.for 5 minutes. Samples were resuspended in 50 μl of FACS staining buffercontaining the appropriate fluorescently-labeled streptavidin. Sampleswere incubated at 4° C. for 30 minutes. Cells were washed with 150 μlFACS staining buffer and centrifuged at 1300 rpm at 4° C. for 5 minutes.This wash step was repeated before samples were resuspended in 250 μl ofFACS staining buffer. Samples were analyzed on a BD LSRFortessa™ cellanalyzer (BD Biosciences) the same day.

Data Analysis

Flow cytometry data was reanalyzed using FlowJo Version 9 software togate specific phenotypic populations. Enumeration of geometric mean wasused to compare VISTA expression in different cell subsets. Eachpopulation was normalized for background by subtracting isotype controlvalues from the mean values of the anti-VISTA treated samples. Graphswere prepared in Prism and statistics were performed using eitherstudent's T-test if only two samples were compared, or one-way ANOVAwith Bonferroni post-tests.

Results:

Expression of VISTA on Human Myeloid and Lymphoid Subsets:

As shown in FIGS. 2A-2E, 3A-3G, 4, 5A-5B and 6A-6C, VISTA expression onCD14⁺ monocytes was significantly different from all other populations(p<0.001). No significant differences between other populations wereseen. Monocytes expressed the highest levels of VISTA in peripheralblood, with the CD14⁺CD16⁻ subset having significantly higher expressionthan CD14^(lo) CD16⁺ cells. While APCs showed moderate expression ofVISTA, lymphoid subsets showed low expression levels.

Expression of VISTA on Human T and NK Subsets:

As shown in FIGS. 7A-7E, 8A-8G and 9, with NK subsets, CD56^(lo) cellsexhibited significantly higher expression levels of VISTA than CD56^(Hi)NK cells. Of T cell subsets, CD8⁺ memory cells expressed the highestexpression levels, although they are not significantly higher than CD8⁺naive or CD4⁺ T cells.

Expression of VISTA on Human Dendritic Cell Subsets:

As shown in FIGS. 10A-10D, 11A-11C and 12, no significant differences inVISTA expression seen; DCs and basophils exhibited low expression ofVISTA, with plasmacytoid dendritic cells (pDCs) generally being higherbut not to a significant extent.

Conclusion: These results show expression of VISTA on various immunecell subsets, and that VISTA is expressed on monocytes most highly, withsome expression on different T cell subsets and NK cells, and little tono expression on B cells.

Example 2: Vista Expression on Peripheral Blood Cells

Methods:

Staining of whole blood: Freshly isolated whole blood (100 μl) wasstained with antibody cocktails as indicated below by incubation for 30minutes at 4° C. Red blood cells (RBCs) were lysed with RBC lysis bufferand the remaining cells were washed 1× with staining buffer. Cells werere-suspended in 200 μl of staining buffer. The data were collected usinga MACSQuant flow cytometer and analyzed using FlowJo analysis software.

Staining of peripheral blood mononuclear cells (PBMCs): Peripheral bloodmononuclear cells were isolated from whole blood using Ficoll gradient.Freshly isolated 1×10⁶ PBMCs were stained with antibody cocktails in 100μl of staining buffer. Samples were incubated for 30 minutes at 4° C.then washed once with staining buffer. Cells were re-suspended in 100 μlof staining buffer. The data were collected using MACSQuant® flowcytometer (Miltenyi Biotec) and analyzed using FlowJo analysis software.

The antibodies used were CD11b, CD33, CD177, CD16, CD15, CD14, CD20,HLADR, CD3, CD4, CD8, CD127, CD69, and FOXP3 antibodies (Biolegend, SanDiego, Calif.). The APC-conjugated mouse anti-human VISTA (clone GG8)was made by ImmuNext (Lebanon, N.H.).

Conclusions:

Expression of VISTA on Healthy Human Peripheral Blood Cells

Whole blood and peripheral blood mononuclear cells were analyzed forVISTA expression using multicolor flow cytometry. As shown in FIGS. 15Aand 15B, the highest level of VISTA expression was detected on monocytesfollowed by neutrophils. Both the CD4+ and CD8+ T cells expressed lowlevel of VISTA as shown in FIGS. 13C and 13D.

Expression of VISTA on Cancer Patient Peripheral Blood Cells

As shown in FIGS. 14A-C, peripheral blood mononuclear cells (PBMCs) fromlung cancer patients were analyzed. FIG. 14A is a representative FACSplot showing analysis of CD14⁺ monocytes and CD15⁺ myeloid derivedsuppressive cells (MDSCs). The results suggest that phenotypically CD15⁺cells are neutrophil derived MDSCs. Additionally, these cells are absentin healthy blood samples. FIG. 14B is a representative histogram ofVISTA expression on healthy and cancer patient derived monocytes,suggesting a higher level of VISTA expression on cancer patient cellscompared to healthy controls. Similarly higher level of VISTA was foundon MDSCs in cancer patients, as shown in FIG. 14C.

FIG. 15A is a representative FACS plot showing the presence ofneutrophil derived MDSCs in the blood of colon cancer patients. FIGS.15B and 15C are representative histograms showing higher level of VISTAexpression on cancer patients' monocytes compared to healthy donor bloodsamples.

Expression of VISTA on Cynomolgus Monkey Peripheral Blood Cells

As shown in FIGS. 16A and 16B flow cytometry analysis of monkey wholeblood revealed the VISTA expression pattern similar to human cells. Bothmonocytes and neutrophils expressed the highest level of VISTA comparedto CD4⁺ (FIG. 16C) and CD8⁺ (FIG. 16D) T cells.

Example 3: Vista Expression in Heme Malignancy Cell Lines at the RNALevel and Protein Level

Because VISTA is expressed in heme malignancies, an anti-VISTA antibodycould potentially target the malignant cells for destruction, as well asblock VISTA and promote anti-tumor immune responses.

The data includes RNAseq analysis of ˜140 heme malignancy cell lines(some cell lines are repeated in the analysis). The data is shown inFIG. 17.

The RNAseq values are listed as FPKM (Fragments Per Kilobase of exon perMillion fragments mapped) values.

In essence, this means that all reads falling in the exonic regions of agene were counted and normalized by both the length of the gene and thetotal number of reads per sample (to account for inter-sampledifferences). The cutoff value is 1; above 1 is positive for VISTAexpression (at the RNA level), below 1 is negative for VISTA expression.

The results indicated that many cell lines are positive at the RNAlevel, primarily acute myeloid leukemias and chronic myelogenousleukemias. This may be expected since VISTA is highly expressed innormal myeloid cells, and because its function is believed to dampenimmune responses, including anti-tumor immune responses.

Example 4: Generation of Monoclonal Antibodies Against Vista

Phage Panning

Twenty four phage panning experiments were carried out to enrich forphage reactive to Cyno VISTA-His. The cynomolgus VISTA protein was usedfor these experiments as it showed better biotin conjugation than thehuman VISTA protein. To determine the success of the phage experiments,phage pools from the individual panning rounds were added to neutravidinplates coated with biotinylated cyno VISTA-His and detected with aHRP-conjugated anti-M13 antibody. Individual colonies were picked fromthe phage selection rounds and Fabs proteins were produced in 96 wellplates. The expressed Fab supernatants were assayed for binding tobiotinylated cyno VISTA-His. This resulted in more than 200 hits.

The VH and VL regions from the Fab plates were amplified, submitted forDNA sequencing and were exported as FASTA files. When picking the clonesthat should be converted and tested as MABs, the clones were chosenbased on sequence diversity as well as having limited post-translationalmodification risks and as few hydrophobic residues as possible.

The VH and VL from the phage clones were sub-cloned into mammalianIgG1/kappa expression vectors and transfected into HEK293 cells. Theantibodies were purified on Protein A Sepharose Fast Flow affinityresin. The concentration of the phage MABs was determined byquantitative ELISA using Nanodrop measurements, The antibody panel wasexpressed at high levels. SDS-PAGE analysis demonstrated the integrityof each expressed antibody variant.

In-line maturation of the phage antibodies was done by amplifying the VHdomains from the polyclonal antibody mixes from the last round ofpanning for cloning into phage vectors that have diversity in the VL.This resulted in an enriched VH pool which was sampled with additionaldiversity in the VL. The phage were taken through 1-2 rounds ofstringent panning with the expectation to identify very high affinitybinders to VISTA ECD His protein. A monoclonal Fab ELISA was run todetermine the success of the maturation. ELISA and expression data wasnormalized to a reference clone set to 100% from the original de novopanning experiment and affinity matured clones with higher bindingsignal to cyno VISTA antigen than the reference clone were identified.This process generated several clones that demonstrated up to 200%binding when screened at low antigen concentration (1 nM), the cloneswith highest affinity were sequenced and produced as MABs.

Hybridoma Generation

One group of BALB/cAnNCrl mice received one intraperitoneal (IP)injection of 50 Hu VISTA-Ig recombinant protein (Sino) emulsified inComplete Freund's Adjuvant followed two weeks later by one IP injectionof 50 μg Hu VISTA-Ig recombinant protein emulsified in IncompleteFreund's Adjuvant. Two weeks later the mice received one IP injection of50 μg cyno VISTA-Fc recombinant protein emulsified in IncompleteFreund's Adjuvant. All mice received a final injection of 25 μg humanand 25 μg cyno VISTA at the base of tail in PBS, five days prior tosplenic harvest for fusion.

Another group of BALB/cAnNCrl mice received one IP injection of 50 μg HuVISTA-His recombinant protein emulsified in Complete Freund's Adjuvant.Two weeks later the mice received one IP injection of 50 μg Hu VISTA-Hisrecombinant protein emulsified in Incomplete Freund's Adjuvant. Twoweeks later the mice received one IP injection of 50 μg Cyno VISTA-Hisrecombinant protein emulsified in Incomplete Freund's Adjuvant. Twoweeks later all mice received a final injection of 25 μg Hu VISTA-Hisand 25 μg Cyno VISTA-His in PBS, three days prior to splenic harvest forfusion.

On the day of fusion, mice were euthanized by CO2 asphyxiation, thespleens were removed and placed into 10 mL of cold phosphate-bufferedsaline. A single cell suspension of splenocytes was prepared by grindingspleens through a fine mesh screen with a small pestle and rinsing withPBS at room temperature. Cells were washed once in PBS and subjected toRBC lysis. Briefly, cells were resuspended in 3 mL of RBC lysis buffer(Sigma #R7757) per every spleen and placed on ice for 5 minutes. Cellswere again washed once in PBS at room temperature and labeled formagnetic sorting. As per manufacturer's instructions, cells were labeledwith anti-murine Thy1.2, anti-murine CD11b and anti-murine IgM magneticbeads (Miltenyi Biotec #130-049-101, 130-049-601 and 130-047-301respectively) then sorted using a MS column with a Midi MACS. Thenegative cell fractions (positive cell fractions were discarded) werefused to FO cells. Fusion was carried out at a 1:1 ratio of murinemyeloma cells to viable spleen cells. Briefly, spleen and myeloma cellswere mixed together, pelleted and washed once in 50 mL of PBS. Thepellet was resuspended with 1 mL of polyethylene glycol (PEG) solution(2 g PEG molecular weight 4000, 2 mL DMEM, 0.4 mL DMSO) per 10e8splenocytes at 37° C. for 30 seconds. The cell/fusion mixture was thenimmersed in a 37° C. water bath for approximately 60 seconds with gentleagitation. The fusion reaction was stopped by slowly adding 37° C. DMEMover 1 minute. The fused cells were allowed to rest for 5 minutes atroom temperature and then centrifuged at 150×g for 5 minutes. Cells werethen resuspended in Medium E-HAT (MediumE (StemCell Technologies cat#03805) containing HAT (Sigma cat #H0262) and seeded in 96-well flatbottom polystyrene tissue culture plates (Corning #3997).

A capture EIA was used to screen hybridoma supernatants for antibodiesspecific for cyno VISTA. Briefly, plates (Nunc-Maxisorp #446612) werecoated at 4 μg/ml for at least 60 minutes with goat anti-mouse IgG (Fc)antibody (Jackson #115-006-071) in coating buffer (Thermo 28382). Plateswere blocked with 200 μl/well of 0.4% (w/v) bovine serum albumin (BSA)in PBS at for 30 minutes at RT. Plates were washed once and 50 μl/wellof hybridoma supernatant was added and incubated at room temperature forat least 30 minutes. Plates were washed once and 50 μl/well of 0.1 μg/mLof cyno VISTA-hulg was added and incubated at RT for 30 minutes. Plateswere washed once and 1:40,000 Streptavidin HRP (Jackson 016-030-084) in0.4% BSA/PBS was added to plates and incubated for 30 minutes at RT.Plates were washed 3× and subsequently developed using 100 μl/well TMBTurbo substrate (Thermo Scientific 34022) incubating approximately 10minutes at RT. The reaction was stopped using 25 μl/well 4N SulfuricAcid and absorbance was measured at 450 nm using an automated platespectrophotometer. Fifteen of the primary hits were selected forsubcloning by limiting dilution and were screened in the same primaryscreen format.

All cyno VISTA reactive hybridoma cell lines were cross screened usinghuman VISTA-Ig to assess cross-reactivity. Briefly, plates(Nunc-Maxisorp #446612) were coated at 4 μg/mL with goat anti-ms Fc(Jackson #115-006-071) in 0.1M sodium carbonate-bicarbonate buffer, pH9.4 (Pierce 28382 BupH™) 0/N at 4° C. Without washing, the wells wereblocked with 200 μl of block (0.4% BSA (Sigma) (w/v) in PBS(Invitrogen)) overnight at 4° C. After removing block solution,undiluted hybridoma supernatants were incubated on coated plates for 30minutes at RT. Plates were washed once with PBST (0.02% Tween 20 (Sigma)(w/v) in PBS), and then incubated for 30 minutes with Hu VISTA-Igdiluted to 100 ng/ml in block. Plates were washed once with and probedwith Goat antihuman-Fc-HRP (Jackson #109-036-098) diluted 1:10,000 inblock for 30 minutes at RT. Plates were again washed and subsequentlydeveloped using 100 μl/well TMB Turbo substrate (Thermo Scientific34022) incubating approximately 10 minutes at RT. The reaction wasstopped using 25 μl/well 4N Sulfuric Acid and absorbance was measured at450 nm using an automated plate spectrophotometer.

Hybridomas that were shown to be reactive to both human and cynomolgusVISTA had their V region antibody sequences cloned. Hybridoma cells wereprepared prior to the reverse transcriptase (RT) reactions withInvitrogen's SuperScript III cells Direct cDNA System. Briefly, theculture medium was discarded and the plate placed on ice and resuspendedin 200 μl cold PBS. Forty microliters was transferred to a MicroAmp fast96 well Reaction PCR plate and the plate was placed on a cold metalplate base, sealed with plastic film and spun at 700 rpm for 3 minutes.The PBS was discarded and to each well, 10 μl Resuspension Buffer and 1μl Lysis Enhancer was added. The plate was sealed and incubated at 75°C. for 10 min and stored at −80° C.

For the RT reaction, each well contained 5 μl water, 1.6 μl 10× DNaseBuffer, 1.2 μl 50 mM EDTA, 2 μl Oligo(dT)20 (50 mM) and 1 μl 10 mM dNTPMix. The plate was incubated at 70° C. for 5 min, followed by incubationon ice for 2 min, then the following reagents were added for each well;6 μl 5×RT Buffer, 1 μl RNaseOUT™ (40 U/μl), 1 μl SuperScript™ III RT(200 U/μl) and 1 μl of 0.1M DTT. The plate was sealed and placed on athermal cycler preheated to 50° C. and incubated at 50° C. for 50minutes, followed by inactivation (5 min incubation at 85° C.). Thereaction was chilled on ice and the single-stranded cDNA was stored at−80° C. until further use.

For V region amplifications, 20 μl PCR reactions were set up. Each wellcontained 16.2 μl water, 2.0 μl 10×PCR Reaction buffer, 0.8 μl MgSO4 (50mM), 0.4 μl 10 mM dNTP, 0.15 μl 100 uM Forward primer mix 0.05 μl 100 uMReverse primer, 0.2 μl HiFi Tag enzyme. The cDNA, prepared as describedabove, was transferred (2 μl/well) to the PCR components mixture, theplate was sealed and an amplification reaction was run; for VH theprogram was (i) 94° C. for 1 min (ii) 94° C. for 15 sec (iii) 55° C. for30 sec (iv) 68° C. for 1 min. Steps (ii-iv) were repeated for a total of35 cycles followed by a final extension at 68° C. for 3 min. for VL theprogram was (i) 94° C. for 1 min (ii) 94° C. for 15 sec (iii) 55° C. for30 sec (iv) 65° C. for 30 sec, (v) 68° C. for 1 min. Steps (ii-v) wererepeated for a total of 35 cycles followed by a final extension at 68°C. for 3 min.

Forward primers were pre-mixed and such mixture was used in ration 3:1with the reverse primer. PCR products were verified on an agarose gel.The reactions were prepared for infusion cloning by the addition ofEnhancer (In-Fusion HC Cloning Kit, cat #639650, Clontech). Fivemicroliters of the PCR reaction was transferred to a PCR plate followedby the transfer of 2 μl of enhancer/well. The plate was sealed andincubated in a thermal cycler (15 min at 37° C. and 15 min at 80° C.).The destination vector (vDR243 or vDR301) was prepared by Esp3Idigestion; (1.5 μg vector was digested in 3 μl Tango Buffer, 21 Esp3Iand water in a 30 μl reaction at 37° C. for 2 hours).

For infusion cloning, 2 μl of enhancer treated PCR product was mixedwith 100 ng Esp3I digested vector and 2 μl of 5× infusion enzyme(Clontech). The infusion reaction was done in 96-well PCR plate format.The plate was incubated for 15 min at 50° C. on a PCR machine and Stellacompetent cells were transformed by heat shock for 40 seconds at 42° C.without shaking and spread on LB agar plates with select antibiotic andincubated overnight at 37° C. Next day, colonies were picked into96-well deep well plates containing LB/Carbenicillin media and grownovernight at 37° C. Frozen stocks were made from overnight culturemixing with equal volume of 30% w/v glycerol. The V regions weresequenced using sequencing primer SPF0052. The sequences were analyzed,one positive well per hybridoma vH and vL was chosen, re-arrayed in newplates and grown overnight in rich medium with ampicillin. Clones thenhad miniprep DNA prepared for small scale transfection in 96-well plate.

Forty eight selected mouse hybridoma sequences for both heavy and lightchain were human framework adapted using an internal software program.One human framework was chosen for each one of the mouse vH or vL. Vregion DNA sequences were obtained through back-translation. SyntheticDNA regions corresponding to the HFA amino acid sequences were orderedfrom Integrated DNA Technologies (Coralville, Iowa). Cloning wasperformed into pre-cut vDR149 and vDR157, human IgG1 and human kapparespectively. Qiagen Endo-free Maxi-prep kits were used to prepare theDNA. Expi293 (100 ml) cultures were used to express this antibody panel.

Example 5: Protocol for Human Vista-Ig T Cell Suppression Assay In Vitro

Mouse A20 cells were stably transfected with either GFP or human VISTA.They were incubated with ova peptide and with DO11.10 T cells. CD25expression by the T cells was measured 24 hours after incubation began.The A20-huVISTA cells suppress CD25 expression by the T cells, but thisreadout is significantly restored by incubation with VSTB95 (FIG. 18).

Example 6: Human Framework Regions Adaptation of Anti-Vista Antibodies

Mouse hybridoma sequences for both heavy and light chain were humanframework adapted by CDR-grafting (Jones, et al. Nature, 321: 522-525(1986) using an internal software program. The program delineates thecomplementarity determining regions (CDRs) of the V region sequencesaccording to the Kabat definitions (Wu, T. T. & Kabat, E. A. (1970). JExp Med, 132, 211-50) and compares the framework regions with the humangermline genes using Blast. The human germline with the highest sequenceidentity to the mouse frameworks was chosen as the acceptor gene forhuman framework adaptation (HFA). In a few cases, closely related humangermline genes were chosen instead, based on previous experience withwell-expressed human frameworks. DNA sequences for the human frameworkschosen for each one of the mouse vH or vL V regions were obtainedthrough back-translation. Synthetic DNA regions corresponding to the HFAamino acid sequences were ordered from Integrated DNA Technologies(Coralville, Iowa). Cloning was performed into human IgG1 and humankappa, respectively.

Example 7: Anti-Vista Antibody Constructs

Plasmid and sequence information for the molecules for cell linedevelopment: Plasmid constructs were generated for anti-VISTA antibodieshaving the VSTB112 variable regions and an IgG1K constant regions(VSTB174, new number due to an allotypic change in the constant region),an IgG2sigma constant region (VSTB140) or an IgG1 protease-resistantconstant region (VSTB149).

Lonza Vectors

The pEE6.4 and pEE12.4 Chinese hamster ovary (CHO) expression vectorsystem (Lonza Biologics, PLC) was established in Biologics Research (BR)and Pharmaceutical Development & Manufacturing Sciences (PDMS) as theprimary expression system for generation of therapeutic mAbs inmammalian expression cell lines. Each vector contains a humancytomegalovirus (huCMV-MIE) promoter to drive the expression of theheavy chain (HC) or light chain (LC) and contains the ampicillinresistence gene. pEE12.4 vector also includes the gene encoding theglutamine synthetase (GS) enzyme. Growth conditions which requireglutamine synthetase activity places selective pressure on the cells tomaintain the expression vector (GS Gene Expression System Manual Version4.0). pEE6.4 was used to clone the HC gene and pEE12.4 to clone the LCgene as single gene vectors. The Lonza double gene plasmid is createdfrom these two Lonza single genes vectors.

Amino Acid Sequences of Variable Heavy ChainRegions of Select VISTA mAbs >VSTB112 heavy chain (SEQ ID NO: 37)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSYGWSYEFDYWGQGTLVTVSS >VSTB50 heavy chain (SEQ ID NO: 38)QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGLNWVRQAPGQGLEWMGWINPYTGEPTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCAREGYGNYIFPYWGQGTLVTVSS >VSTB53 heavy chain (SEQ ID NO: 39)QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYTIHWVRQAPGQGLEWMGYIIPSSGYSEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAYDDYYDYYAMDYWGQGTLVTVSS >VSTB95 heavy chain (SEQ ID NO: 40)EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYGMSWVRQAPGKGLEWVASIISGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIYDHDGDYYAMDYWGQGTTVTVSS Amino Acid Sequences of Variable Light ChainRegions of Select VISTA mAbs >VSTB50 light chain (SEQ ID NO: 41)DIVMTQTPLSLSVTPGQPASISCRASESVDTYANSLMHWYLQKPGQPPQLLIYRASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQTNEDPRTFGQGTKLEIK >VSTB53 light chain (SEQ ID NO: 42)DIVMTQSPLSLPVTPGEPASISCRSSQTIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQASHVPWTFGQGTKLEIK >VSTB95 light chain (SEQ ID NO: 43)DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGQGTKLEIK >VSTB112 light chain (SEQ ID NO: 44)DIQMTQSPSSLSASVGDRVTITCRASQSIDTRLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSAYNPITFGQGTKVEIK >VSTB116 light chain (SEQ ID NO: 45)DIQMTQSPSSLSASVGDRVTITCRASQSINTNLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQARDTPITFGQ GTKVEIK

Example 8: ELISA and FACS Screening of Anti-Vista Antibodies

These experiments were to determine the ability of the producedantibodies to bind human or cynomolgus VISTA protein in an ELISA, aswell as to determine, using FACS screening, the ability of theantibodies to bind VISTA protein on the surface of K562 cells (humanmyelogenous leukemia cell line) expressing human or cynomolgus VISTAproteins.

Methods:

ELISA procedure summary: Plates were coated overnight at 4° C. with 1μg/ml SB0361 (human) or SB0361 (cyno (cynomolgus)) proteins, which arethe extracellular domains of VISTA from the respective species.Antibodies were diluted to 1 μg/ml as a starting concentration with 1:4step-wise dilutions for a total of 4 concentrations and incubated atroom temperature room temperature (RT) for 2 hours. Mouse anti-humanIgG1-HRP (horseradish peroxidase) was used for detection and incubatedfor 1 hour at RT. All washes were performed using PBS-Tween (0.05%).

FACS procedure summary: 2×10⁵ K562-G8 (human) or K562-C7 (cyno) cellswere stained with 5 μg/ml of each test antibody and incubated for 30minutes at 4° C. Goat anti-human IgG1-PE (phycoerythrin) antibody wasused as a secondary detection antibody at 5 μg/ml. Cells were run on aBD Fortessa and analyzed using FlowJo software (Tree Star, Inc.,Ashlang, Oreg.) for MFI (mean fluorescence intensity) of the livepopulation.

Data Analysis/Results: For each antibody, a subjective score (Yes/No)was given relating to whether the antibody bound robustly or not forboth the ELISA and FACS analysis for each of the 4 assays. If anantibody gave a “No” result for binding in either assay, it was repeatedto confirm that it was negative. The results are shown in Table 7 belowand in FIGS. 19A-19F and 20A-20F.

TABLE 7 INX Code Hu ELISA Cyno ELISA Hu FACS Cyno FACS 1 Y Y Y Y 2 Y Y YY 3 Y Y Y Y 4 Y Y Y Y 5 Y Y Y Y 6 Y Y Y Y 7 Y Y Y Y 8 Y Y Y Y 9 Y Y Y Y10 Y Y Y Y 11 N N N N 12 Y Y Y Y 14 Y Y Y Y 16 Y Y Y Y 17 Y Y Y Y 18 Y YY Y 19 Y Y Y Y 20 Y Y Y Y 21 Y Y Y Y 22 Y Y Y Y 23 N N N N 24 N N N N 25Y Y Y Y 26 N Y N Y 28 Y Y Y Y 30 N N N N 31 N N N N 32 N N N N 33 Y Y YY 34 Y Y Y Y 35 Y Y Y Y 36 Y Y Y Y 37 Y Y Y Y 38 Y Y Y Y 39 Y Y N N 40 YY Y Y 41 Y Y Y Y 42 Y Y Y Y 43 Y Y Y Y 44 Y Y Y Y 45 Y Y Y Y 46 Y Y Y Y47 Y Y Y Y 48 Y Y Y Y 49 Y Y Y Y

Example 9: Screening Results of Anti-Human Vista Antibodies Using theMixed Lymphocyte Reaction (MLR) and Staphylococcus Enterotoxin B (SEB)Activation Assays

The purpose of this study was to present data supporting theidentification of multiple functional α-VISTA antibodies that enhancecellular immune responses in the mixed lymphocyte reaction (MLR) assay,as well as the staphylococcus enterotoxin B activation (SEB) assay.

The mixed lymphocyte reaction (MLR) is a standard immunological assaythat depends upon MHC class I and II mismatching to drive an allogeneicT cell response. Peripheral blood mononuclear cells are isolated fromtwo mismatched individuals, incubated together and as a result of thesemismatches, proliferation and cytokine production occurs.

Material and Methods:

10% AB Media was prepared by combining 500 ml of RPMI with 50 ml ofhuman AB serum, 5 ml of Penicillin/Streptomycin (10,000 U/ml), 5 ml ofL-glutamine (100×) and 10 ml of HEPES (1M). Media was stored for nolonger than 14 days. 1 mCi tritiated thymidine was prepared by diluting0.2 ml of thymidine stock (1 mCi/ml) in 9.8 ml of RPMI.

Soluble VISTA antibodies were diluted to 20 μg/ml in 10% AB serum media.100 11.1 of the appropriate antibody solutions was added to theappropriate wells of a 96 well U-bottom plate (Falcon product #353077 orequivalent). After the various cellular populations were added, thefinal concentration was 10 μg/ml.

Isolation of white blood cells: Donors were at least 18 years of age,generally healthy and selected randomly from the local population.Transferred donor blood from isolation tubes to 50 ml conicals.Under-laid 15 ml of Ficoll 1077 per 25 ml of blood being careful not tomix with the blood. Centrifuged the cells at 1250 g for 25 minutes atroom temperate with no brake. White blood cells were isolated at theinterphase of the Ficoll and the serum and diluted the cells into 40 mlof Hanks Balances Salt Solution (HBSS). Centrifuged the cells at 453 g(1500 rpm) for 10 minutes at 4° C. Resuspended the cells in 50 ml ofHBSS and counted by transferring 500 μl to a separate tube.

Mixed lymphocyte reaction (MLR) 96 well plate setup: Determined theappropriate number of “stimulator cells” and “responder cells” neededfor the assay based on the number of samples to be analyzed. Thestimulator population is seeded at 0.5×10⁵ cells/well and the responderpopulation is seeded at 1.0×10⁵ cells/well of a 96 well U-bottom plate.All conditions must be performed in triplicate. The appropriate numberof “stimulator cells” were pipetted into a new conical and centrifugedas previously described. Resuspended cells in 10 ml and irradiated with4000 rads. Centrifuged cells as previously described and resuspended ata concentration of 1×10⁶/ml in 10% AB serum media and added 50 μl toappropriate wells. Isolated the required number of responder cells andcentrifuged as previously described and resuspended at a concentrationof 2×10⁶/ml in 10% AB serum media and added 50 μl to appropriate wells.Incubated the cells for 5 days at 37° C. and 5% CO₂. On the fifth day,removed 30 μl of supernatant for analysis of interferon gamma (IFN-γ)production. On the fifth day, added 25 μl of a 40 μCi/ml tritiatedthymidine solution to each well and incubated for 8 hours at 37° C. and5% CO₂. Transferred cells to the 96 well micro scintillation plate permanufacturer's instructions. Counted using the micro scintillationcounter per manufacturer's instructions. IFN-γ concentration wasdetermined by ELISA (eBioscience cat #88-7316-88) using manufacturer'sprotocol.

Data analysis: Calculated the average counts per minute (CPM) or IFN-γconcentration for the non-treated wells. Calculated the average CPM orIFN-γ for each of the test groups. Log₁₀ transform the data set. Using12 MLR fold-scores for each compound, calculated the average for the setof 12 test groups of each compound Average score for 12experiments=Σ[(log₁₀ (Average CPM of triplicate for testcompound))−(log₁₀ (Average CPM of triplicate for No Treatment))]/12

Acceptance criteria: All test reagents and appropriate controls weretested for endotoxin prior to running the assay and have levels of <0.1EU/mg. The responder cells alone had CPM counts below 700 CPM on averageindicating that the cells were quiescent when incubated alone. The CPMfor the MLR group was at least 2 fold higher than the CPM for respondercells incubated alone indicating that a reaction had occurred and thatthe donors are a mismatch. All MLR assays included a human IgG1 negativecontrol protein. The result of the human IgG1 negative control was notstatistically different from the non-treated samples based upon use of astudent's t-test.

Screening of anti-VISTA antibodies in the MLR: Initial screen of allcompounds. Prior to running the MLR with the anti-VISTA antibodies,antibodies were confirmed to bind both cell bound VISTA via FACSanalysis and VISTA protein via ELISA. Antibodies S26 (VSTB77), S30(VSTB86), S31 (VSTB88), S32 (VSTB90) and S39 (VSTB74) failed thisinitial screen but were still tested in the assay. For the purpose ofinitial screening, all antibodies were tested at 10 μg/ml in the MLRwith proliferation and IFN-γ being the parameters measured (FIGS.21A-21D and 22A-22B).

Selection of six lead antibodies. From the initial screen, sixcandidates were chosen for further analysis: VSTB112 (S2), VSTB116 (S5),VSTB95 (S16), VSTB50 (S41), VSTB53 (S43) and VSTB60 (S47).

Dilution studies of the top six candidates in the MLR: Protocoladjustments. The protocol is identical as previously described with theadjustment that antibodies were diluted to the following concentrations:30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0 μg/ml.

Determination of IC₅₀ values: Raw CPM counts and IFN-γ concentrationswere used to determine the IC₅₀ for each of the antibodies. Calculationsof IC₅₀ were determined through use of the program “EZ-R stats.” Sixindividual responders were used to determine the IC₅₀ values. IndividualCPM counts and IFN-γ concentrations in the MLR with dose titrations ofthe lead candidates.

TABLE 8 IC₅₀ values for both CPM and IFN-γ in the MLR VSTB112 VSTB116VSTB95 VSTB50 VSTB53 VSTB60 (S2) (S5) (S16) (S41) (S43) (S47) CPM −0.67−0.78 −0.54 −0.12 −0.33 0.02 Gamma −0.42 −0.16 0.22 0.06 0.27 0.4 **Values are in log₁₀ of antibody concentrations.

Conclusion: The initial screen indicated that multiple VISTA specificantibodies were capable of enhancing the MLR cellular immune response.Antibodies were then ranked based upon efficacy and variance and basedupon these results, VSTB112, VSTB116, VSTB95, VSTB50, VSTB53 and VSTB60were chosen to evaluate in dose-titration experiments. VSTB60 induced aweaker response than the other five antibodies in the dose-titrationexperiments.

The staphylococcus enterotoxin B (SEB) activation assay: SEB is abacterial super-antigen that induces activation of specific Vβ+ T cells.Peripheral blood mononuclear cells are isolated and incubated with theSEB antigen in culture, which induces robust cytokine production. Thisassay was conducted on the five lead candidates.

Preparation of 10% AB Media, preparation of 1 mCi tritiated thymidine,preparation of soluble VISTA antibodies, and isolation of white bloodcells were all performed as previous described above in the MLR.

SEB 96 well plate setup: Determined the appropriate number of respondercells needed for the assay based on the number of samples to beanalyzed. The responder population is seeded at 2.0×10⁵ cells/well of a96 well U-bottom plate. All conditions must were performed intriplicate. Centrifuged cells as previously described and resuspended ata concentration of 4×10⁶/ml in 10% AB serum media and added 50 μl to theappropriate wells. Added 50 μl of 10% AB serum media containing the SEBantigen at a concentration of 40 ng/ml. In the described experiments,SEB was obtained from Sigma Aldrich (cat #S0812). The finalconcentration in the well was at 10 ng/ml. Incubated the cells for 3days at 37° C. and 5% CO₂. On the third day, removed 30 μl ofsupernatant for analysis of IFN-γ production. Added 25 μl of a 1 mCi/mltritiated thymidine solution to each well and incubated for 8 hours at37° C. and 5% CO₂. Cells were transferred to the 96 well microscintillation plate per manufacturer's instructions. Counted using themicro scintillation counter per manufacturer's instructions. IFN-γconcentration was determined by ELISA (eBioscience cat #88-7316-88)using manufacturer's protocol.

Protocol: Data analysis. Calculated the average counts per minute (CPM)or IFN-γ concentration for each of antibodies at all concentrations.Acceptance criteria were performed as previously described.Determination of IC₅₀ values was performed as described. Individual CPMcounts and IFN-γ concentrations in the SEB assay with dose titrations ofthe lead candidates.

TABLE 9 IC₅₀ values for both CPM and IFN-γ in the SEB. VSTB112 VSTB116VSTB95 VSTB50 VSTB53 VSTB60 (S2) (S5) (S16) (S41) (S43) (S47) CPM −1.16−1.44 −1.12 −0.74 −1.06 not done Gamma −1.24 −0.35 0.05 1.69 −1.05 notdone **Values are in log10 of antibody concentrations.

Conclusions: VISTA specific antibodies enhanced cytokine production andproliferation in a dose dependent manner in the SEB assay. IC₅₀ valuesfrom the SEB study were generally similar to the results from the MLRdilution studies.

Example 10: Epitope Binning Assay

Methods: ProteOn XPR36 system (BioRad) was used to perform epitopebinning.

ProteOn GLC chips (BioRad, Cat #176-5011) were coated with two sets of 6monoclonal antibodies (mAbs) using the manufacturer instructions foramine-coupling chemistry (BioRad, cat #176-2410).

Competing mAbs were pre-incubated in excess (250 nM final concentration)with human VISTA (25 nM final concentration) for 4 hours at roomtemperature and 6 at a time were run over the chip coated with thepanels of coated mAbs with an association time of 4 minutes followed bydissociation for 5 minutes. Following each run, the chips wereregenerated with 100 mM phosphoric acid.

The data analysis involved grouping all sensorgrams by ligand andapplying an alignment wizard, which automatically performs an X and Yaxis alignment, and artifact removal. An Interspot correction was thenapplied to the data.

A non-competing mAb was defined as having a binding signal the sameor >A1 signal (binding to human VISTA only).

A competing mAb was defined as having binding signal <<A1 signal (i.e.,binding to human VISTA only).

Results: In the example sensorgram shown in FIG. 23, theVSTB85 antibodywas coated on the Proteon SPR chip and VISTA protein preincubated withthe indicated competitors was run over the chip. VSTB50 is an example ofa non-competitive antibody, as a positive response was seen when theVISTA/VSTB50 complex was run. GG8, VSTB49 and VSTB51 complexed withVISTA did not bind to the VSTB85 coated on the chip and were thereforeclassified as competing for the same binding site on VISTA as VSTB85.

TABLE 10 Sample Set #1: coupled to sensor Sample Set #2: coupled tosensor L1 L2 L3 L4 L5 L6 L1 L2 L3 L4 L5 L6 Samples Group GG8 B85 B95B104 B112 B113 B50 B53 B66 B67 IE8 B116 GG8 1 Y Y Y Y Y Y N Y N Y Y YVSTB100.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB101.001 1 Y Y Y Y Y Y N Y N YY Y V5TB102.001 1 Y Y Y Y Y Y N Y N Y Y Y V5TB103.001 1 Y Y Y Y Y Y N YN Y Y Y V5TB104.001 1 Y Y Y Y Y Y N Y N Y Y Y V5TB105.001 1 Y Y Y Y Y YN Y N Y Y Y V5TB106.001 1 Y Y Y Y Y Y N Y N Y Y Y V5TB107.001 1 Y Y Y YY Y N Y N Y Y Y V5TB108.001 1 Y Y Y Y Y Y N Y N Y Y Y V5TB109.001 1 Y YY Y Y Y N Y N Y Y Y VSTB110.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB111.001 1Y Y Y Y Y Y N Y N Y Y Y VSTB112.001 1 Y Y Y Y Y Y N Y N Y Y YVSTB113.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB114.001 1 Y Y Y Y Y Y N Y N YY Y VSTB115.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB116.001 1 Y Y Y Y Y Y N YN Y Y Y VSTB49.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB51.001 1 Y Y Y Y Y Y NY N Y Y Y VSTB53.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB59.001 1 Y Y Y Y Y YN Y N Y Y Y VSTB65.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB67.001 1 Y Y Y Y YY N Y N Y Y Y VSTB70.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB81.001 1 Y Y Y YY Y N Y N Y Y Y VSTB92.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB95.001 1 Y Y YY Y Y N Y N Y Y Y VSTB97.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB98.001 1 Y YY Y Y Y N Y N Y Y Y VSTB99.001 1 Y Y Y Y Y Y N Y N Y Y Y VSTB50.001 2 NN N N N N Y N Y N N N VSTB54.001 2 N N N N N N Y N Y N N N VSTB56.001 2N N N N N N Y N Y N N N VSTB60.001 2 N N N N N N Y N Y N N N VSTB63.0012 N N N N N N Y N Y N N N VSTB66.001 2 N N N N N N Y N Y N N NVSTB73.001 2 N N N N N N Y N Y N N N VSTB76.001 2 N N N N N N Y N Y N NN VSTB78.001 2 N N N N N N Y N Y N N N VSTB84.001 2 N N N N N N Y N Y NN N VSTB85.001 3 Y Y Y Y Y Y N Y N Y 1 Y VSTB74.001 4 N N N N N N N N NN N N IE8 5 Y 1 Y Y Y Y N Y N Y Y Y mAb immobilized on sensor Y = Yescompeted (signal << than A1-human VISTA only) N = No competed (signal >than A1-human VISTA only) I = Inconclusive (signal similar to A1-humanVISTA only)

Example 11: Proteon Affinity Determination

Antibodies were captured on ProteOn chips using anti-IgG Fc coatedsurfaces. The antibodies were tested for binding of human and cynomolgus(cyno) VISTA extracellular domains (ECDs) at concentrations of VISTAproteins ranging from 0.39 nM to 100 nM. The antigens were allowed tobind/associate to the antibody-coated chips for 4 minutes after whichtime dissociation was monitored for 30 minutes. Chips were regeneratedwith two treatments of 100 mM phosphoric acid for 18 seconds. Allexperiments were run at 25° C. and data was fit to 1:1 Langmuir bindingmodel.

Example 12: Effects of Anti-Vista Treatment in a Mb49 Murine BladderTumor Model

Methods:

C57Bl/6 mice were injected with MB49 tumor cells. Once the tumors wereestablished, anti-VISTA treatment was initiated. Tumor growth was thenmonitored 3 times/week. Mice were euthanized, in accordance with IACUCregulations, once the tumors reached 15 mm in any dimension.

For each experiment, a frozen vial of MB49 cells was thawed and grown inRPMI 1640 (+L-Glut) with 10% serum and penicillin/streptomycinantibiotics. After three days in culture, the cells were harvested usingStemPro Accutase and resuspended in RPMI at a concentration of 5×10⁶cells/ml and 50 μl injected per mouse.

Female C57Bl/6 mice, aged 6-8 weeks were purchased from the NationalCancer Institute. Upon arrival they were allowed to acclimatize for oneday prior to having their right flanks shaved and their tails tattooed.They were then injected three-five days later.

Tumor Injection (Intradermal): Mice were injected intradermally (i.d.)on their shaved flank with 50 μl of MB49 cell suspension (250,000cells).

Monitoring Tumor Growth: Tumor growth was measured using electroniccalipers first across the widest dimension (L) and secondly at a 90°angle to the first measurement (W). Tumor volume derived as follows:

Volume=(L ² *W ²)/2

Tumors were considered established once they reached ˜5 mm in diameter(˜60 mm³ volume). Once established, treatment was initiated. Tumorgrowth was measured three times per week over the course of treatmentand until the experiment was terminated.

Anti-VISTA Treatment: Chimerized 13F3-mIgG2a monoclonal antibody wasinjected intraperitoneally at 10 mg/kg. Injection schedules were thriceweekly for four weeks.

Euthanizing Mice: As per IACUC requirements, animals were euthanizedonce their tumors reached 15 mm in the longest dimension.

Analyzing Efficacy: Mouse tumor volumes were analyzed using Excel fordata management, and GraphPad Prism for graphing. Statistical analysiswas performed using a macro for R statistical computing software.

The experimental design is shown in FIG. 24.

Results:

Ch13F3-mIgG2a treatment in female mice led to complete tumor rejection(CR) in 70% of the animals and partial remission (PR) in 30% (n=7)(Table 13 and FIG. 25B). In contrast, all of the control mIgG2a-treatedmice showed progressive growth of the tumors (6/6)(FIG. 25A). These datademonstrate that anti-VISTA treatment can have a profound effect ontumor growth.

TABLE 11 Complete remission (CR) versus partial remission (PR) Female13F3 IgG2a (n = 7) CR 5 PR 2 till day 32

The human VISTA sequence is shown in FIGS. 26 and 27, adapted from Wanget al., 2011, supra, the contents of which are incorporated herein intheir entirety.

Example 13: Epitope Mapping of Anti-Vista Antibodies UsingHydrogen/Deuterium (H/D) Exchange Studies

To identify the epitopes for VSTB50, 60, 95 and 112 on human VISTA,solution hydrogen/deuterium exchange-mass spectrometry (HDX-MS) wasperformed using the corresponding Fabs. For H/D exchange, the proceduresused to analyze the Fab perturbation were similar to that describedpreviously (Hamuro et al., J. Biomol. Techniques 14:171-182, 2003; Hornet al., Biochemistry 45:8488-8498, 2006) with some modifications. Fabswere prepared from the IgGs with papain digestion and Protein A captureusing Pierce Fab Preparation Kit (Thermo Scientific, Cat #44985). Thehuman VISTA protein sequence contains six N-linked glycosylation sites.To improve the sequence coverage, the protein was deglycosylated withPNGase F. The deglycosylated VISTA protein was incubated in a deuteratedwater solution for predetermined times resulting in deuteriumincorporation at exchangeable hydrogen atoms. The deuterated VISTAprotein was in complex with either Fab of VSTB50, VSTB60, VSTB95 orVSTB112 in 46 μL deuterium oxide (D₂O) at 4° C. for 30 sec, 2 min, 10min and 60 min. The exchange reaction was quenched by low pH and theproteins were digested with pepsin. The deuterium levels at theidentified peptides were monitored from the mass shift on LC-MS. As areference control, VISTA protein was processed similarly except that itwas not in complex with the Fab molecules. Regions bound to the Fab wereinferred to be those sites relatively protected from exchange and, thus,containing a higher fraction of deuterium than the reference VISTAprotein. About 94% of the protein could be mapped to specific peptides.

The solution HDX-MS perturbation maps of VISTA with VSTB50/VSTB60, andVSTB95/VSTB112 are shown in FIG. 28 top and bottom, respectively. Twoepitope groups were identified. Anti-VISTA VSTB50 recognizes the sameepitope as VSTB60 does; VSTB95 binds to another epitope region asVSTB112 does on VISTA. Anti-VISTA VSTB50 and 60 share the same epitopewhich comprises segments, ₁₀₃NLTLLDSGL₁₁₁ (SEQ ID NO:62), and₁₃₆VQTGKDAPSNC₁₄₆ (SEQ ID NO:63) (FIG. 28 top). Anti-VISTA VSTB95 and112 appear to target similar epitopes, comprising segments₂₇PVDKGHDVTF₃₆ (SEQ ID NO:75), and ₅₄RRPIRDLTFQDL₆₅ (SEQ ID NO:65) (FIG.28 bottom). There are two other segments showing weak perturbation byVSTB95 and 112, including residues 39-52 and 118-134. However, thelevels of the reduction are not as strong as the previous regions (27-36and 54-65) in the differential map. Although one peptide, ₁₀₀TMR₁₀₂showing strong perturbation by VSTB95 and 112, is located on the otherface of VISTA surface, it is distant from the epitope regions, 27-36 and54-65. This perturbation could be due to allosteric effect. These HDX-MSresults provide the peptide level epitopes for anti-VISTA antibodies.There were no overlapping epitope regions for these two epitope groups.These results are in agreement with the previous competition binningdata in that they do not compete with each other.

Example 14: Structure Determination of the Human Vista ECD:VSTB112 FABComplex by Protein Crystallography

In an effort to determine the VISTA structure and to delineate theepitope and paratope defining the interaction between VISTAextracellular domain (ECD) and the Fab fragment of lead antibodyVSTB112, the complex was crystallized and structure determined to 1.85 Åresolution. The structure of the ECD of human VISTA in complex with theFab fragment of the antibody VSTB112 was determined in an effort both todetermine the structure of VISTA ECD itself and to define theepitope/paratope for this interaction. The structure reveals VISTA toadopt an IgV fold with a chain topology similar to the TCR Vα chain. Inaddition to the canonical disulfide bond bridging B and F strands in theback and front faces of the β-sandwich, the structure reveals the ECD tohave two additional disulfide bonds, one tethering the CC′ loop to thefront sheet and a second between the A′ and G′ strands. Although crystalcontacts between VISTA molecules are present, they are minor and thereis no evidence for a dimer of VISTA ECDs based on this structure. TheVSTB112 epitope is shown to comprise the portions of the VISTA BC, CC′,and FG loops together with residues of the front beta sheet (C′CFG)nearest those loops. The paratope is biased largely toward heavy chaininteractions with CDR L3 making minimal contact.

Epitope/Paratope Defining VISTA:VSTB112 Interaction

VSTB112 Fab buries a surface area of 1024.3 Å2 upon binding VISTA ECD,with burial of the heavy chain surface accounting for 715.3 Å2 of thistotal. Seven hydrogen bonds and 4 salt bridge interactions are formedbetween VISTA and VSTB112 light chain and 10 hydrogens and 2 salt bridgeinteractions between VISTA and VSTB112 heavy chain. VSTB112 recognizesresidues in the front sheet strands C′, C, F, and G on the ends proximalto the FG loop as well as residues in the BC, FG, and CC′ loops (FIGS.29 and 30). Interactions with the CC′ loop account for most of thecontacts with the Fab light chain with only residues E125 and R127 inthe FG loop making additional light chain interactions. Residues 119 to127 corresponding to the VISTA FG loop account for 38% of the total1034.8 Å2 of surface area buried upon binding VSTB112. Notably, thisloop is highly polar, comprised of the following sequence—IRHHHSEHR-(SEQ ID NO:76). Additionally, W103 in the VSTB112 CDR H3 packs nicelyagainst the backbone of VISTA residues H122 and H123, and VISTA H121makes an edge on interaction with the aromatic ring of F55 in CDR H2.

A comparison of epitope regions identified by crystallography and HDX isshown in FIG. 31.

Example 15: Activation of T Cells and Monocytes by Anti-Vista Antibodies

The functional effect of anti-VISTA antibodies was evaluated in two invitro assays, mixed leukocyte reaction (MLR) and SEB (Staphylococcusenterotoxin B). Both assays measure T cell proliferation and cytokineinduction as their primary readouts, but these effects are due todifferent mechanisms. In the MLR, peripheral blood mononuclear cells(PBMCs) from two different human donors are incubated together, andmajor histocompatibility complex (MHC) mismatch between T cells of onedonor and dendritic cells of the other donor results in T cellproliferation and interferon (IFNγ) production. In the SEB assay, PBMCsfrom a single donor are incubated with a bacterial superantigen, whichdirectly links MHC Class II protein on the surface of antigen-presentingcells (APC) to the T-cell receptor (TCR) on T cells, causing T cellactivation, proliferation, and cytokine secretion. In both assays,VSTB112, which is the parent molecule of VSTB174, demonstrateddose-dependent induction of T cell proliferation and cytokineproduction, and was most potent among the candidates (FIGS. 21A-21D,Table 12).

TABLE 12 EC50 values for the MLR assay readouts. VSTB112 (parent ofVSTB174) was the most potent molecule. EC₅₀ proliferation EC₅₀ IFNγproduction Candidate (μg/ml) (μg/ml) VSTB112 0.21 0.38 VSTB116 0.17 0.69VSTB95  0.29 1.67 VSTB50  0.77 1.14 VSTB53  0.47 1.88 VSTB60  1.04 2.48

Monocyte Activation Assays

The assay data, shown in Table 12, was generated with VSTB112, theparent molecule of VSTB174. To better understand the activity ofVSTB174, monocyte activation assays were conducted. The results showedthat incubation of VSTB174 with whole PBMCs induced upregulation ofactivation markers (CD80 and HLA-DR) on CD14+ monocytes, indicating aneffect of antibody binding to an immune cell subset known to expres highlevels of VISTA (FIG. 32). A further question is whether the effects onmonocyte activation in whole PBMC could be facilitated by any antibodythat binds VISTA and has an IgG1 Fc. Antibodies VSTB103 and VSTB63 bindto VISTA with high affinity (KD 6.36E-10 and 8.30E-10 respectively) andto cells expressing VISTA protein, similar to VSTB112 and VSTB111.VSTB103 is in the same epitope bin as VSTB112, while VSTB63 is in adifferent epitope bin; neither antibody facilitated monocyte activation.Taken together, these results show that one mechanism by which VSTB174may exert its effect on T cell activation/proliferation is via monocyteactivation facilitated by NK cells.

Preparation of Media

500 ml of RPMI 1640 (Corning, 10-040-CV) was combined with 50 ml ofhuman AB serum (Valley Biomedical, Inc, Lot #3C0405), 5 ml ofPenicillin/Streptomycin (Lonza, 17-602E) 10,000 U/ml, 5 ml ofL-glutamine (100×) (Gibco, 25030-081) and 10 ml of HEPES (1M) (FisherBP299-100, Lot #-1). Media was stored for no longer than 14 days at 4°C.

Preparation of soluble VISTA and control antibodies

Antibodies were diluted to 2× desired concentration in 10% AB serummedia: VSTB174: lot VSTB174.003

Added 100 μl of the appropriate antibody solutions to the appropriatewells of a 96 well U-bottom plate (Falcon, 353077). After the variouscellular populations were added in 100 the final concentration of eachantibody was 1, 0.1 or 0.01 g/ml. IgG1 control antibody CNTO 3930 (Lot6405, ENDO<0.1 EU/mg) was added at a final concentration of 1 μg/ml.

The PBMCs were isolated

Donors were at least 18 years of age, generally healthy and selectedrandomly from the local population.

Donor blood was transferred from isolation tube to 50 ml conicals.

15 mls of Ficoll 1077 (SIGMA, 10771) were under-laid being careful notto mix with the blood. This was per 25 mls of blood.

The cells were centrifuged at 1250 g for 25 minutes at room temperaturewith no brake.

The white blood cells were isolated at the interphase of the Ficoll andthe serum and the cells were diluted into 40 ml of Hanks Balanced SaltSolution (HBSS).

The cells were centrifuged at 453 g (1500 rpm) for 10 minutes at 4° C.

The cells were resuspended in 50 mls of HBSS and were counted bytransferring 500 l to a separate eppendorf tube.

Additionally, a Pan Monocyte isolation kit from Miltenyi was used permanufacturer's instructions (cat #130-096-537) to isolate CD14+ cells bynegative selection in several treatment groups.

In Vitro Culture Setup

The appropriate number of cells needed was determined for the assaybased on the number of samples to be analyzed. The responder populationwas seeded at 2.0×10⁵ cells/well of a 96-well U-bottom plate. For theCD14 negatively selected population, 0.5×10⁵ cells were seeded. Allconditions were performed in triplicate.

The cells were centrifuged as described above and resuspended at aconcentration of 2×10⁶/ml for the whole PBMC population and 0.5×10⁶/mlfor the CD14 negatively selected population in 10% AB serum media andadded 100 l of test antibody to appropriate wells bringing the totalvolume in each well to 200 1.

The cells were incubated for 1, 2, or 3 days at 37° C. and 5% CO₂.

Antibody Staining and Flow Cytometry

The 96 well U-bottom plate was centrifuged for 5 minutes at 453 g andremoved the supernatant.

Cells were washed with 200 μl PBS and centrifuged as in step 5.5.1.

The supernatant was discarded and resuspended in 50 μl of PBS containingthe following antibodies:

-   -   CD14-APC (clone HCD14) 1:250 (Biolegend cat #325608)    -   HLA-DR-PE Cy7 (clone L243) 1:250 (Biolegend cat #307616)    -   CD80-PE (clone 2D10) 1:250 (Biolegend cat #305208)    -   Hu FcR binding inhibitor (eBioscience cat #14-9161-73)

Was incubated for 20 minutes on wet ice in the dark.

150 μl of PBS was added and centrifuged as in step 5.5.1.

150 l of PBS buffer was added and analyzed via FACS.

Samples were run on a Miltenyi MACSQuant 10-parameter flow cytometer andanalyzed using FlowJo 9.7.5 for expression of HLA-DR and CD80 on theCD14+ population. Geometric mean fluorescence intensity (MFI), astatistic that defines the central tendency of a set of numbers, wasused as the defining statistic to compare treatments.

Statistical Analysis

All statistics were carried out in Prism GraphPad, version 6. Pair-wisecomparisons amongst the groups were made at each of the time-pointsusing One-Way ANOVA with Tukey correction for multiplicity. P-valuesless than 0.05 for all tests and comparisons were deemed significant.For all graphs and tables, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Example 16: ADCC and ADCP Activities of Anti-Vista Antibodies

VSTB174 has an IgG1 Fc, which can confer antibody-dependentcell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediatedphagocytosis (ADCP) activity. Both types of assays were conducted andshowed that VSTB174 could lyse or phagocytose K562-VISTA cells (FIGS.33-34), but not K562 myeloma cell line parental cells (data not shown).An additional mechanism of action of VSTB174 to modulate the inhibitoryaction of VISTA may be the lysis or engulfment of cells expressing highlevels of VISTA, thus removing them from the local microenvironment.

Example 17: ADCP Activities of Additional Anti-Vista Antibodies

An in vitro phagocytosis assay was used to study the enhancement ofmacrophage-mediated phagocytosis of cells ectopically expressing VISTAby anti-human VISTA mAbs (VSTB173 and VSTB174). These mAbs were clonedinto different Fc backbones (IgG1 WT (wild type), IgG1 PR (proteaseresistant), and IgG2σ) and were postulated to potentially have differentactivities with respect to enhancing phagocytosis. The IgG1 and IgG1 PRbackbones are capable of binding to Fc receptors and have the potentialto cause ADCP, while the IgG2σ does not bind to Fc receptors and shouldnot mediate ADCP.

Anti-VISTA antibodies were tested in ADCP assays with K562 parental andK562-VISTA target cells. As shown in FIGS. 35-36, VSTB174, VSTB149,VSTB173 and VSTB145 enhanced hMac phagocytosis of K562-VISTA cells.VISTA antibodies VSTB140 or VSTB132, with the IgG2σFc that did not bindFc receptors, did not enhance phagocytosis as expected. VISTA mAbsVSTB174 and VSTB173 with IgG1 Fc showed more robust phagocytosis thanVSTB149 and VSTB145 with the IgG1PR Fc (see Tables 13 and 14 for EC₅₀values).

TABLE 13 Anti-human VISTA mAb EC₅₀ values. Treatment VSTB174 VSTB149VSTB140 EC₅₀ 0.0782 0.1142 NA

TABLE 14 Anti-human VISTA mAb EC₅₀ values. Treatment VSTB173 VSTB145VSTB132 EC₅₀ 0.0146 0.1075 NA

VSTB174 and VSTB173 showed weak enhancement of phagocytosis of K562parental cells at the highest concentration (FIGS. 35-36), which may bedue to low expression of VISTA by the K562 cells. The other anti-VISTAantibodies did not enhance phagocytosis of the K562 cells.

The negative control antibodies were each tested at two differentconcentrations in the K562-VISTA phagocytosis assay, but did not induceany phagocytosis. This result indicates that the phagocytosis mediatedby the anti-VISTA antibodies is specific and due to VISTA antigenexpression by the K562-VISTA cells.

Example 18: ADCC Activities of Additional Anti-Vista Antibodies

In order to test their ability to induce ADCC, the following three humananti-VISTA antibodies were tested:

VSTB174 (IgG1)

VSTB149 (IgG1 PR)

VSTB174.LF (IgG1 LF (low fucose)).

Each antibody was tested at six different concentrations within the sameplate, in triplicate over two separate experiments for a total of sixdata points.

VSTB174, VSTB149, and VSTB174.LF each demonstrated measurable ADCCactivity at 10, 1, 0.1 and 0.01 μg/mL, while only the LF antibodydemonstrated measurable ADCC activity at 0.001 μg/mL; none of theantibodies demonstrated ADCC at 0.0001 μg/mL. As each of theseantibodies has an IgG1 or IgG1 variant Fc, this result is expected. TheLF antibody demonstrated increased ADCC potency as evidenced by thesmaller EC₅₀ value for the LF antibody curve (0.002293 μg/mL) ascompared to the regular IgG1 antibody curve (0.02381 μg/mL). The IgG1 PRantibody curve had an EC₅₀ value similar to the regular IgG1 curve(0.01846 μg/mL).

TABLE 15 EC₅₀ values (μg/mL) of three tested anti-VISTA antibodies asdetermined by ADCC analysis. anti-VISTA Antibody EC₅₀ (μg/mL) VSTB174(IgG1) 0.02381 VSTB149 (IgG1 PR) 0.01846 VSTB174.LF (IgG1 LF) 0.002293

The human IgG1, human IgG1 PR and human IgG1 LF antibodies all showedmeasurable ADCC mediated killing at the 10, 1, 0.1 and 0.01 μg/mLantibody concentrations, while only the LF antibody showed killing atthe 0.001 μg/mL antibody concentration. None of the anti-VISTAantibodies showed killing at the 0.0001 μg/mL antibody concentration.

The LF antibody showed approximately 10 times more potent ADCC killingthan either the regular IgG1 antibody or the IgG1 PR antibody, as seenin the EC50 values.

Example 19: Affinity of Vstb174 for Human and Cynomolgus Vista

The affinity of VSTB174 for human and cynomolgus monkey VISTAextracellular domain (ECD) was determined by surface plasmon resonance(SPR) methods on a ProteOn instrument. VSTB174 displayed very similar KDvalues for each protein, 1.56E-10 M for human VISTA ECD and 8.66E-11 Mfor cynomolgus VISTA.

Example 20: Vista Antibodies Exhibit Efficacy in Murine Tumor Models

Mouse Strains, Reagents and Tumor Models

For the in vivo studies, human VISTA knockin (VISTA-KI) miceback-crossed onto a C57Bl/6 background were used.

An anti-human VISTA antibody was generated to enable testing in theVISTA-KI mice, using the VSTB174 variable region grafted onto mouse FcIgG2a (VSTB123).

The MB49 bladder cancer was evaluated in the VISTA KI mice,

In addition to published studies demonstrating that anti-VISTA antibodytherapy inhibits tumor growth in wild type mice (Le Mercier et al.,2014), anti-tumor efficacy has been demonstrated with the surrogatehamster antibody in wt mice using different dosing schedules, and in theVISTA-KI mice treated with VSTB123.

In Vivo Efficacy Studies in the MB49 Tumor Model in VISTA-KI Mice

MB49 efficacy studies were conducted in female VISTA-KI mice, testingVSTB123 at several doses ranging from 1-10 mg/kg. Mice were injectedintradermally with 250,000 MB49 tumor cells on day 0. On day 6, dosingbegan as indicated in FIG. 37 (either 10 mg/kg of the isotype controlmIgG2a, or the indicated doses of VSTB123; 10 mice/group).

VSTB123 was more effective at higher vs lower doses, as shown in FIG.37. Doses of 10 mg/kg and 7.5 mg/kg were equivalent, while tumors grewmore quickly in the mice dosed at 5 or 1 mg/kg.

Example 21: Detection of Vista Expression in Human Tumors withAnti-Vista Antibodies

FIG. 1 shows VISTA expression by an AML tumor cell line—this and the RNAseq expression data in FIG. 17 support the idea that VISTA is expressedby AML cells and that anti-VISTA drug be efficacious through directlytargeting these cells for immune modulation or antibody-mediatedkilling.

Data to evaluate VISTA expression in lung cancer was obtained from lungtumor samples from surgical resections. Cells were dissociated andcharacterized for expression of VISTA and many other markers. Resultsshowed that 13/13 lung tumors (squamous or adenocarcinomas) containedCD14+ VISTA+ myeloid cells, (FIG. 38).

Example 22: Detection of Vista Expression in Lung Tumors UsingAnti-Vista Antibodies

An immunohistochemistry assay was developed using clone GG8, ananti-human VISTA mouse IgG1. This mAb was used to investigate thestaining of VISTA in non small cell lung cancer (NSCLC) FFPE tumorsections.

FFPE tumor sections were treated with standard antigen retrieval methodsprior to staining. GG8 mouse anti-human VISTA antibody was used at a1:500 dilution. GG8 binding was detected using a rabbit anti-mousepolyclonal antibody, followed by anti-rabbit polymer HRP. Counterstainwith hematoxylin followed, then tumor sections were scored.

VISTA expression in lung cancer was mostly restricted to the immuneinfiltrate (example shown in FIG. 39) and high levels of VISTA positivecells were present in many lung cancer samples

Example 23: Structure of the Extracellular Domain (Ecd) of Human Vistain Complex with the Fab Fragment of VSTB174

VISTA antigen variants were generated and purified for crystallography.Recombinant his-tagged VSTB174 Fab was internally expressed andpurified. Crystals were generated and used to collect higher resolutiondata for the VISTA ECD:VSTB174 Fab complex using synchrotron radiationand the structural determination was solved using combinations ofhomology modeling and electron density analyses (FIG. 29(Top)).

The structure of the VISTA ECD:VSTB174 Fab complex was determined byx-ray crystallography to a resolution of 1.85 Å, providing the firststructure of the VISTA ECD and delineating the VSTB174 epitope andparatope. The VISTA ECD adopts an IgV fold with a topology similar toCTLA-4 ECD, but possesses a unique G′ strand that extends the frontsheet of the β-sandwich. A′ and G′ are further tethered chemically via adisulfide bridge formed between residues C12 in the A′ strand and C146in the G′ strand. Six cysteines were found to be engaged in threeintramolecular disulfide bonds, and, based on crystal contacts, there isno evidence for a dimeric VISTA.

VSTB174 recognizes residues in the front sheet strands C′, C, F, and Gon the ends proximal to the FG loop as well as residues in the BC, FG,and CC′ loops.

Example 24: Monocyte Activation by Anti-Vista Antibodies Requires Cd16(FcγRIII) Cross-Linking

The present study was designed to evaluate the ability of anti-VISTAantibodies to activate monocytes in culture. Monocyte activation wasassessed by the upregulation of surface expression of canonical markersof monocyte activation: CD80, CD86, HLA-DR, and PD-L1. Given thatVS7113174 has an active Fc, the roles of CD16 and other Fe receptors inanti-VISTA-mediated monocyte activation were studied. In particular, theability of anti-CD16, anti-CD32, and anti-CD64 antibodies in blockinganti-VISTA-mediated monocyte activation was examined, in which VSTB112(HuIgG1 active Fc) or VSTB140 (IgG2sigma silent Fc) was used to elicitmonocyte activation. Soluble Fc fragments were also used in the sameassay to block Fc binding non-specifically.

CD16 expression on PBMC (which lack neutrophils) is typically restrictedto NK cells and inflammatory monocytes (CD14+/−, CD16+). NK cells weredepleted to determine their role in anti-VISTA induced monocyteactivation. Additionally, IFN-γ, a major product of activated NK cellscapable of monocyte activation was blocked to assess its individualcontribution.

Methods

Preparation of Media

All dilutions and culturing were done in RPMI (Life Technologies; cat#11875-091) containing 10% Human AB serum (Sigma-Aldrich, Cat #H5667)and 1% Penn/Strep (Life Technologies; cat #15140-122).

Blocking Antibodies

Anti-CD16 (Biolegend Cat #302050, Clone 3G8), anti-CD32 (BD Cat #552930,Clone FLI8.26), and anti-CD64 (Biolegend Cat #305016, clone 10.1) werediluted to 20 μg/ml in complete media as indicated. The in-house block(IHB) was diluted to 8 mg/mL, also in complete medium.

50 μl of these stocks were plated out in triplicate on a 96 well platefor each activating/blocking condition indicated. 50 μL of mediacontaining no antibodies was used for the “no block control”.

Cell Preparation

2 vials of frozen PBMC (HemaCare PBOO9C-1, 10×10⁶ cells per vial) pereach donor indicated were thawed in a 37 degree water bath, diluted to10 mL in complete media, and centrifuged @1500 RPM for 5 minutes.

The media containing diluted freezing media was removed and cells werewashed with 10 mL of fresh media and spun down as above. Prior to thisspin, a 10 μL sample was retained for each sample.

Cell counts were determined by diluting samples 1:2 in trypan blue andcounting on a Countess™ Automated Cell Counter (Cat #C10227). Cells wereresuspended in complete media at a concentration of 2×10⁶ cells/mL. 100μL of this cell preparation was added to all experimental wells. Thecell/blocking antibody mixture-containing plates were incubated for 15minutes at 37 degrees C.

Activation

VSTB112, VSTB140, and HuIgG1 isotype control (11.76 mg/mL) were alldiluted to 20 μg/mL in complete media. After completion of theincubation step, 50 μL of the activating antibody stocks were added tothe appropriate well containing cells and blocking reagents. Finalconcentration of blocking and activating antibodies was 5 μg/mL. Finalconcentration of RIB was 2 mg/mL. Cells were incubated at 37 degrees C.overnight (20 hours).

Analysis

After incubation, all experimental plates were spun down at 1500 RPM for5 minutes. 150 μL of the medium was removed via pipetting and stored at−80° C. for later analysis. 150 μL of FACS buffer (Becton Dickinson; cat#554657) was added to each well, mixed by pipetting, and samples werespun down once more at 1500 RPM for 5 minutes. Cells were resuspended in50 uL of FACS buffer containing 2 mg/mL of the IHB and were incubated at4 degrees C. in the dark for 20 minutes. After incubation, 50 μL of thefollowing staining mix diluted in FACS buffer was added to all samples(all antibodies diluted 1:25): CD14—APC (Biolegend, clone HCD14);CD80-PE (Biolegend, clone CD10); CD86-FITC (Biolegend, clone IT2.2);HLA-DR—APCCy7 (Biolegend, clone L243); PD-L1-PeCy7 (Biolegend, clone29E2A3). Samples were incubated for 30 minutes at 4 degrees C. in thedark. Following incubation, 100 μL of FACS buffer was added to each welland plates were spun down @1500 RPM for 5 minutes. Cells were washed 1×as described above and finally resuspended in 200 μL of FACS buffercontaining Aqua LIVE/DEAD® (LifeTechnologies, Cat #L34957) per themanufacturer's instructions. Samples were run on a BD FACS Cantor™ IIand analyzed with FlowJo ver9.2.

Results

After the completion of culture, viable CD14+ cells were analyzed forexpression of CD80, CD86, HLA-DR, and PD-L1 (FIG. 40, showing PD-L1expression). Compared to monocytes in PBMC cultures that were treatedwith the HuIgG1 isotype control, monocytes in cultures treated withVSTB112 had increased levels of each of these markers. The level of thisincrease varied from donor to donor (data not shown). Monocytes incultures treated with VSTB140 did not show elevated levels of activatingmarkers indicating that an active Fc domain is required for the effect(FIG. 40). Further, the addition of competitive binding inhibitor Fcfragments (MB) to the cultures muted and, in some cases, completelyabrogated VSTB 112-mediated activation. This effect was not CD32 orCD64-dependent as antibodies blocking these receptors did not blockVSTB112-mediated monocyte activation (data not shown).

CD16 expression on PBMC (which lack neutrophils) is typically restrictedto NK cells and inflammatory monocytes (CD14+/−CD16+). Preliminary datahad identified a role for NK cells in the in vitro activation ofmonocytes by anti-VISTA antibodies. Depletion of NK cells or blockade ofthe IFNγ receptor significantly reduced the extent of anti-VISTA inducedmonocyte activation, suggesting that both NK cells and IFNγ contributeto anti-VISTA-mediated monocyte activation in vitro (data not shown).

In summary, using CD80, CD86, HLA-DR, and PD-L1 as markers of monocyteactivation, the ability of VSTB112 (parent molecule of antibody VSTB174)and its silent Fc derivative, VSTB140, to activate monocytes in PBMCcultures was compared with a non-specific human IgG1 control antibody(ONTO 3930). By all parameters, monocytes were activated by VSTB112 andnot by VSTB140. This activity appears to be Fc dependent due to theinability of VSTB140 to activate and the ability of soluble Fc fragmentsto partially mute the activating capabilities of VSTB112 (FIG. 40,showing PD-L1 as marker of monocyte activation). Additionally, thisactivation was dependent on the presence of NK cells in these culturesand on antibody-induced production of IFN-γ as removal of either ofthese components from the in vitro culture system markedly attenuatedmonocyte activation (data not shown). As shown herein, the mechanism ofactivation appears to be dependent on crosslinking of the Fc, receptorCD16, as it can be completely and robustly mimicked by the addition ofantibodies that crosslink CD16, even in the absence of VISTA-bindingantibodies.

Example 25: Tumor Growth Inhibition by Anti-Vista Antibody RequiresEffector Function

VISTA, a negative regulator of T cells, is expressed on manyhematopoietic cells and highly expressed in the tumor microenvironment(Le Mercier, et al., Cancer Research 74(7):1933-44, 2014). Treatment ofmice bearing tumors with an anti-mouse VISTA antibody in vivo results insignificantly reduced tumor growth (Le Mercier, et al.).

This study was conducted to determine the effect of anti-human VISTAantibodies VSTB123 and VSTB124 on the growth of established MB49 tumorsin male or female hVISTA KI (knock-in) mice. These mice have the humanVISTA cDNA knocked-in in place of the mouse VISTA gene, and express onlyhuman VISTA both at RNA and protein level. MB49 tumor cells express maleH-Y antigen (Wasiuk et al., Cancer Immunol Immunother 61:2273-82, 2012),a self-antigen in male mice but a foreign antigen in female mice.Treatment was initiated when tumors reached 3-5 mm in diameter.Anti-VISTA or control antibodies were dosed 3 times per week at 10 and 5mg/kg for ten doses. All mouse groups were evaluated for tumor volume,survival, weight and changes in immune populations in peripheral bloodduring the course of the experiment. Drug pharmacokinetics (PK) andanti-drug antibody (ADA) development were also evaluated.

Methods

Study Design

Parallel and identical studies were conducted in male and female hVISTAKI mice. For each gender, the mice were divided in 5 groups of 6-7 micetreated respectively with VSTB123 or VSTB124, either at 10 or 5 mg/kg,or mIgG2a control antibody at 10 mg/kg. See FIG. 41 for experimentaldesign.

Cell Source and Preparation

The MB49 cells were confirmed to be free of mycoplasma and othercontaminants (IMPACT™ SC testing at IDDEX RADIL Case #22209-2014). Onecell vial was thawed and grown in RPMI 1640 (+L-Glut) with 10% FBS andpen/strep antibiotics. After three days in culture, cells were harvestedby brief incubation with StemPro® Accutase®, washed twice andresuspended in cold RPMI at 5×10⁶ cells/ml prior to injection into themice. All culture reagents were purchased from Gibco and Hyclone.

Test Agents and Dosage

VSTB123 and VSTB124 are chimeric anti-human VISTA antibodies made byJanssen. Each has the same anti-human VISTA variable region, derivedfrom VSTB174, but cloned into a mouse IgG2a Fc (VSTB123) or a mouseIgG2a ala/ala silent Fc (VSTB124). Antibodies and mIgG2a (BioXcellBE0085, clone C1.18.14 lot #5035/0514) controls were diluted in PBS andadministered by intraperitoneal injection in a volume of 0.2 ml todeliver a dose of 10 or 5 mg/kg.

Mice

hVISTA KI mice were bred at Sage Labs (Boyertown, Pa.). The mice, aged8-12 weeks, first transited for 3 weeks in the quarantine facility, andthen were transferred to the regular facility. They were acclimated for2 days prior to having their right flanks shaved and their tailstattooed. Tumor cells were injected 5 days later.

Intradermal Cell Injection

Mice were injected intradermally in their shaved right flank with 50 μlof MB49 cell suspension (˜250,000 cells). All mice in which theinjection went poorly (leak from injection site or subcutaneousinjection instead of intradermal) were removed from the experiment.

Randomization, treatment initiation and tumor measurement

Tumors were measured on day 4 post-injection in most male mice, whentumors had reached a diameter between 3 mm and 5 mm. Based on theobservation that most mice showed evidence of tumor growth bymeasurement or visual inspection, the mice were randomly assigned totreatment groups. Treatment was initiated on day 5. Tumor growth wasmonitored 2-3 times a week over the course of treatment and until theexperiment was terminated. The formula (L×W²)/2 was used to determinetumor volume (L is the length or longest dimension, and W is the widthof the tumor).

Partial remission (PR) was reached when tumor was half (or more reducedin size but greater than 13.5 mm³) of the initial volume for 3consecutive measurements. Complete remission was reached when any tumorwas less than 13.5 mm³ for 3 consecutive measurements.

Results

As shown in FIG. 42A (left), female mice treated with VSTB123 at 10mg/kg showed significant reductions in tumor volume as compared to thecontrol group. The effect on tumor growth could be detected as early asday 13, after 3 doses of antibody. In addition, some mice in eachVSTB123 treatment group showed complete and durable tumor regressions:5/7 mice in the 10 mg/kg group and 3/6 mice in the 5 mg/kg group. Noneof the 6 control group mice regressed (data not shown).

VSTB124 treatment did not inhibit tumor growth in females at either dose(FIG. 42A, right). As VSTB123 has a mouse IgG2a that can bind to Fcreceptors, while VSTB124 has a silent Fc, this result suggests that Fcbinding is important for efficacy in this model.

The mice were monitored for survival for 52 days (FIG. 42B). For femalemice, survival comparisons were more difficult because two of sixcontrol animals were still alive at 52 days. However, 7/7 mice treatedwith VSTB123 at 10 mg/kg were alive at day 52 (p=0.0108), while 4/6 micetreated with VSTB123 at 5 mg/kg were still alive at that day. Treatmentof female mice with VSTB124 did not result in survival improvement.

As demonstrated herein, treatment with VSTB123 in female hVISTA KI micebearing MB49 tumors led to complete remission (CR) in 85% (5/7 mice) ofthe group at 10 mg/kg, with a significant increase in survival(p=0.0108); VSTB123 treatment at 5 mg/kg led to CR in 3 out of 6 mice(50%). In contrast, VSTB124 did not significantly affect tumor growth orsurvival. This result, compared with results of VSTB123, suggests thatefficacy with anti-VISTA antibody in the MB49 model may require anactive Fc.

Example 26: VSTB174 Triggers Release of Cytokines

Anti-VISTA antibodies induce activation of CD14+ monocytes in whole PBMCcultures, as measured by upregulation of costimulatory markers such asCD80 and HLADR. The present study determined changes, if any, incytokines in human PBMC cultures cultured with anti-VISTA antibodyVSTB174.

Monocytes are innate leukocytes that represent ˜10-30% of the peripheralblood isolated by Ficoll density centrifugation. Monocytes have beenshown to play important roles in both inflammatory and anti-inflammatoryresponses based upon the level of costimulatory proteins and cytokinesthey express. Anti-VISTA antibodies (including VSTB174) activate CD14+monocytes in whole PBMC cultures as measured by upregulation ofcostimulatory markers on the cell surface, such as CD80 and HLA-DR. Todetermine whether anti-VISTA antibody treatment would alter theproduction of cytokines in the assay, whole PBMCs were treated for 24hours with VSTB174 and the supernatants were analyzed by Luminex® forthe differential expression of 41 cytokines.

As shown herein, culture of the anti-human VISTA antibody VSTB174 withwhole PBMC resulted in significantly increased expression of manycytokines in human PBMCs in vitro.

Methods

Preparation of Media

Combined 500 ml of RPMI 1640 (Corning, 10-040-CV) with 50 ml of human ABserum (Valley Biomedical, Inc., Lot #3C0405), 5 ml ofPenicillin/Streptomycin (Lonza, 17-602E) 10,000 U/ml, 5 ml ofL-glutamine (100×) (Gibco, 25030-081) and 10 ml of HEPES (1M) (FisherBP299-100, Lot #-1). Media was stored for no longer than 14 days at 4°C.

Preparation of Anti-VISTA VSTB174 and Control Antibodies

Antibodies were diluted to 2× desired concentration in 10% AB serummedia. Added 100 μl of the appropriate antibody solutions to theappropriate wells of a 96 well U-bottom plate (Falcon, 353077). Afterthe cells were added in 100 μl, the final concentration of each antibodywas 10, 1, 0.1 or 0.01 μg/ml. IgG1 control antibody CNTO 3930 (Lot 6405,ENDO<0.1 EU/mg) was added at a final concentration of 10 μg/ml. Eachcondition was run in triplicate.

Isolation of PBMCs

Donors were at least 18 years of age, generally healthy and selectedrandomly from the local population. Three donors provided PBMCs for thisstudy. Transferred donor blood from isolation tube to 50 ml conicals.Under-laid 15 mls of Ficoll 1077 (SIGMA, 10771) being careful not to mixwith the blood. This was per 25 mls of blood. Cells were centrifuged at1250 g for 25 minutes at room temperature with no brake. Isolated thewhite blood cells at the interphase of the Ficoll and the serum anddiluted the cells into 40 ml of Hanks Balanced Salt Solution (HBSS).Cells were centrifuged at 453 g (1500 rpm) for 10 minutes at 4° C. Cellswere resuspended the cells in 50 mls of HBSS and counted by transferring500 ?alto a separate Eppendorf tube.

In Vitro Culture Setup

The appropriate number of cells needed for the assay was determinedbased on the number of samples to be analyzed. The PBMCs were seeded at2.0×10⁵ cells/well of a 96-well U-bottom plate. All conditions wereperformed in technical triplicates.

Cells were centrifuged at 453 g (1500 rpm) for 10 minutes at 4° C. andresuspended at a concentration of 2×10⁶/ml in 10% AB serum media andadded 100 μl to appropriate wells bringing the total volume in each wellto 200 μl. Cells were incubated for 24 hours at 37° C. and 5% CO₂.Collected 100 μl of supernatant for analysis by Luminex®.

Multiplex Analysis

Cytokines were measured using Millipore Human cyto/chemo MAG Premix 41Plex kit (Cat #HCYTMAG-60K-PX41, EMD Millipore Corporation, Billerica,Mass.). Calibration curves from recombinant cytokine standards wereprepared with three-fold dilution steps in the same matrix as thesamples. High and low spikes (supernatants from stimulated human PBMCsand dendritic cells) were included to determine cytokine recover,Standards and quality controls were measured in technical triplicate,each triplicate test sample was measured once, and blank values weresubtracted from all readings. All assays were carried out directly in a96-well filtration plate (Millipore, Billerica, Mass.) at roomtemperature and protected from light. Briefly, wells were pre-wet with100 μl PBS containing 1% BSA, then beads, together with a standard,sample, spikes, or blank were added in a final volume of 100 andincubated together at room temperature for 30 minutes with continuousshaking. Beads were washed three times with 100 μl PBS containing 1% BSAand 0.05% Tween 20, A cocktail of biotinylated antibodies (50 μl/well)was added to beads for 30-minute incubation at room temperature withcontinuous shaking. Beads were washed three times, then streptavidin-PEwas added for 10 minutes. Beads were again washed three times andresuspended in 125 μl of PBS containing 1% BSA and 0.05% Tween 20. Thefluorescence intensity of the beads was measured using the Bio-Plex®array reader. Bio-Plea® Manager software with five parametric-curvefitting was used for data analysis.

Statistical Analysis

All statistics were carried out in R Statistical Computing Language.Cytokine concentration values below detection (<OOR) were rescaled tothe lowest detectable concentration, and values above accuratequantitation (>OOR) were rescaled to the maximum linearly quantifiableconcentration. Statistical outliers were removed prior to statisticalanalysis on the basis of Grubbs' p<0.05 and outlier distance of greaterthan 1 standard deviation from the group mean using a single step.Pair-wise comparisons amongst the groups were made at each of thetime-points using One-Way ANOVA with Tukey Honest SignificantDifferences. P-values less than 0.05 for all tests and comparisons weredeemed significant. For all graphs and tables, *p<0.05, **p<0.01,***p<0.001, ****p<0.0001. FIG. 43 was produced with completehierarchical clustering of cytokines using the heatmap.2 function in theg-plots package in R.

Results

To determine effects of anti-VISTA antibodies on whole PBMCs, VSTB174was added to cell cultures at different concentrations for 24 hours, andsupernatants were analyzed for levels of 41 cytokines. Whole PBMCstreated with VSTB174 had statistically significant increases in theexpression of a large number of cytokines, many of which are canonicallyexpressed by monocytic and granulocytic cells (FIG. 43). Each donor PBMCresponse was unique in the intensity of the response driven by VSTB174.Donors 1 and 2 showed significant increases in many more analytes thanDonor 3 did (FIG. 43). Also, when all three donors showed significantincreases in a particular analyte, the fold change over baseline wasusually lowest for Donor 3 (data not shown).

Effects of VSTB174 were most likely to be detected when the drug waspresent between 0.1-10 μg/ml, with few analytes significantlyupregulated at 0.01 μg/ml (FIG. 43).

The cytokines significantly elevated over the IgG1 control for alldonors were the following: IL-6, TNFα, MCP-3, MDC, MIP-1β, IP-10,IL-1Rα, GM-CSF, IL-12p70 and GRO.

Some cytokines were significantly elevated only in Donors 1 and 2:MIP-1α, IL-1β, RANTES, G-CSF, IL-1α, IL-7, IL-12p40, IL-13, IFNγ, TNFβ,IFNα (elevated in donor 3 but still close to baseline), IL-4, IL-10,FGF-2, fractalkine, VEGF, IL-17, Flt3L, IL-9, TGFα, IL-15, EGF, andPDGF-αα.

Two cytokines, MCP-1 and IL-8, were significantly elevated only in Donor3. The baseline levels of these cytokines were above the range thatcould be quantitated in the assay for Donors 1 and 2, so it is possiblethat the analytes became elevated with VSTB174 treatment, but it was notmeasurable (listed as OOR>). The baseline and treatment levels of MCP-1and IL-8 were within the dynamic range of the assay for Donor 3.

There were several cytokines that did not change compared to baseline inany donor with VSTB174 treatment: sCD40L, eotaxin, IL-5, PDGF-ββ, IL-2,IL-3. IL-2, IL-3 and sCD40L were significantly elevated in Donor 1 withdrug treatment, but the levels of IL-2 and IL-3 still remained very low.sCD40L only became elevated in Donor 1 at the 0.1 μg/ml dose, and not atany other doses.

As demonstrated herein, VSTB174 induced enhanced production of manycytokines from PBMCs in multiple human donor samples, in adose-dependent fashion.

Further, in vivo studies in female hVISTA KI mice also showed increasedproduction of proinflammatory cytokines (e.g., MCP-1, IP-10, IL-8, IL-6,MIP-1b, IL-10, IL-7, IFN-γ, G-CSF, RANTES, IL-15, TNFα, IL-1β, MIP-1a,IL-1a, GM-CSF, IL-12p40, IL-13, and/or eotaxin) in response to VSTB123.In particular, the upregulated cytokines have been shown to be involvedin recruitment, migration or activation of myeloid cells. Both hVISTA KImice implanted with MB49 tumor cells as well as naïve hVISTA KI miceshowed similar cytokine release profiles (data not shown).

Example 27: Vstb123 Induces Migration of Cd80+ Macrophages to the TumorEnvironment

VISTA is a negative regulator of T cells that is expressed on mosthematopoietic cells. The present study was conducted to identify changesin immune population numbers and activation phenotype in MB49tumor-bearing hVISTA KI mice in response to treatment with anti-humanVISTA VSTB123 or VSTB124 antibodies. The hVISTA KI mice have the humanVISTA cDNA knocked-in in place of the mouse VISTA gene, and werepreviously confirmed to express only human VISTA both at RNA and proteinlevel. MB49 tumor cells express male H-Y antigen, a self-antigen in malemice but a foreign antigen in female mice and highly expressed in thetumor microenvironment. As shown herein, mice with tumors responded withincreased myeloid infiltration after treatment with either VSTB123 orVSTB124, and increased expression of CD80 activation marker ontumor-infiltrating macrophages with VSTB123 treatment.

Methods

Study Design

The hVISTA KI mice were divided into 3 groups of 5 female mice each.Each mouse was injected with MB49 tumor cells in the right flank on day0. At day 7, 9, and 11, mice were injected with 10 mg/kg of mIgG2acontrol antibody, VSTB123, or VSTB124. At day 12, mice were euthanizedand blood, spleens, and tumors were analyzed by multiple parameters.FIG. 44A illustrates this experimental design.

Mice

The hVISTA KI mice are bred at Sage Labs (Boyertown, Pa.). The mice,aged 8-12 weeks, first transited for 3 weeks in the quarantine facility,and then were transferred to the regular facility. They were acclimatedfor 2 days prior to having their right flanks shaved and their tailstattooed. Tumor cells were injected 5 days later.

Cell Source and Preparation

The MB49 cells were confirmed to be free of mycoplasma and othercontaminants (IMPACT™ SC testing at IDDEX RADIL Case #22209-2014). Onecell vial was thawed and grown in RPMI 1640 (+L-Glut) with 10% FBS andpen/strep antibiotics. After three days in culture, cells were harvestedby brief incubation with StemPro® Accutase®, washed twice andresuspended in cold RPMI at a concentration of 5×10⁶ cells/ml, and 50 μl(2.5×10⁵ cells) injected per mouse. All culture reagents were purchasedfrom Gibco and Hyclone.

Intradermal Cell Injection

Mice were injected intradermally in their shaved flank with 50 ml ofMB49 cell suspension (˜250,000 cells). All mice in which the injectionwent poorly (leak from injection site or subcutaneous injection insteadof intradermal) were removed from the experiment.

Test Agents and Dosing

VSTB123 and VSTB124 were generated by Janssen. VSTB123 is an anti-humanVISTA antibody comprised of the VSTB174 variable region on a muIgG2a Fcscaffold. VSTB124 is an anti-human VISTA antibody comprised of theVSTB174 variable region on a muIgG2a Fc scaffold with ala/ala mutationsthat silence the Fc. Control mouse antibody (mIgG2a) was generated byBioXcell, clone C1.18.4, Lot #5386-2/1014, 8.4 mg/ml 1× in PBS.

Antibodies were diluted in PBS to 1 mg/ml for dosing. Mice were injectedintraperitoneally with a volume of 0.2 ml, to deliver a finalconcentration of 10 mg/kg. As outlined in FIG. 44A, mice receivedantibody therapy on days 7, 9, and 11 post tumor injection.

Tissue Harvest

Mice were euthanized using CO₂ in compliance with the Dartmouth IACUCprotocol. Mice were exsanguinated by cardiac puncture and bloodcollected. Spleen and tumors were dissected.

Spleens were dissociated using collagenase (1 mg/ml, Sigma Aldrich) inHBSS and the gentle MACS™ dissociator (Miltenyi Biotec) in accordancewith the manufacturer's instructions. Cells were passed through a 40 μmfilter, then subjected to red blood cell lysis in 3 ml ACK lysis buffer(Lonza, Lot No. 0000400419) for 5 minutes. After 1 wash in HBSS, cellswere resuspended in PBS and immunostained.

Tumors were dissociated using the Tumor Dissociation Kit and the gentleMACS™ dissociator (Miltenyi Biotec), following manufacturerinstructions. Following dissociation, cells were passed over a 40 μmfilter, then subjected to red blood cell lysis using ACK lysis buffer.After 1 wash in HBSS, cells were resuspended in a tracked volume of PBSand immunostained.

Single cell suspensions from the draining lymph node were prepared bymechanical disruption and passage through a 40 μm filter. Cells werewashed, counted, and resuspended in RPMI.

Blood samples were spun down to separate plasma and whole blood cells.Plasma was collected and subsequently frozen and kept at −80° C. to beused for cytokine and ANA ELISA analysis. Whole blood cells weresubjected to red blood cell lysis in 3 ml ACK lysis buffer (Lonza, LotNo. 0000400419) for 5 minutes. After 1 wash in HBSS, cells wereresuspended in PBS and used for immunostaining.

Flow Cytometry

Single-cell suspensions were Fc-blocked with anti-murine CD16/32(Miltenyi) (1:200) for 15 minutes at 4° C. Cells were incubated withantibody cocktails diluted in PBS for 30 minutes. After 2 washes in PBS,cells were resuspended in Fix/Permeabilization working solution(eBioscience) for 30 minutes on ice. Cells were spun, supernatantdiscarded and resuspended in PBS and incubated overnight.

Myeloid Panel:

-   -   Live/Dead Yellow (Life Technologies) (1:1000)    -   Ly6G-FITC (Biolegend, 1A8) (1:200)    -   CD45-PE (Biolegend, 30-F11) (1:800)    -   CD80-PE/CF594 (BD Biosciences, 16-10A1) (1:200)    -   Ly6C-PerCP/Cy5.5 (Biolegend, HK1.4) (1:100)    -   CD11c-PE/Cy7 (Biolgend, N418)(1/200)    -   MHC class II-Alexa Fluor 647 (Biolegend, M5/114.15.2) (1/400)    -   CD86-Alexa Fluor 700 (Biolegend, GL-1) (1/200)    -   F4/80-APC/Cy7 (Biolegend, BM8) (1/200)    -   CD11b-BrV421 (Biolegend, M1/70) (1/100)

Cells were rinsed and run on a Gallios 10-color flow cytometer. Allcells were gated on live/dead and positive CD45 staining. Granulocyteswere CD11b+, Ly6G+ and Ly6C−. Monocytes were CD11b+, Ly6G−, and Ly6C+.Macrophages were CD11b+, Ly6G−, Ly6C−, and F4/80+. Dendritic cells weregated on Ly6G−, Ly6C−, CD11c+ and were CD11b medium or high. Flowcytometry data were analyzed using FlowJo.

Statistical Analysis

All statistics were carried out in Prism GraphPad, version 6. For eachcondition, values were compared using ANOVA with Tukey correction. Inall cases comparisons were made to the mIgG2a control-treated animals.Significance is summarized by asterisks using GraphPad standards, withone asterisk indicating *P<0.05, two ** indicating P<0.01, three ***indicating P<0.001, and four **** indicating P<0.0001.

Results

Tumors dissected from the flanks of anti-VISTA or controlantibody-treated animals were analyzed to determine effects uponcellular populations by flow cytometry. Tumor-infiltrating macrophageswere examined for anti-VISTA induced changes in expression of activationmarkers CD80, CD86, and MHC class II. FIG. 44B illustrates the resultsof the flow cytometry analysis. Tumor-infiltrating macrophages,regardless of treatment, expressed higher levels of CD86 than didsplenic macrophages (data not shown), but did not further upregulateCD86 as a function of anti-VISTA treatment. Conversely, CD80 expressionwas significantly increased on tumor-infiltrating macrophages of hVISTAKI mice treated with VSTB123, but not on those treated with VSTB124(FIG. 44B).

Example 28: Vstb123 Induces Migration of MPO+ Cells to the TumorMicroenvironment

The present study was conducted to identify changes, if any, in thetumor environment in MB49 tumor-bearing hVISTA KI mice in response totreatment with anti-human VISTA VSTB123 or VSTB124 antibodies. As shownherein, VSTB123 induced migration of myeloperoxidase-stained cells tothe tumor environment.

Methods

Tumors and spleen were rapidly dissected after death from MB49tumor-bearing hVISTA KI mice that were treated as described in Example27. Tumor and spleen samples were then put into cassettes and fixed for2-3 weeks (and typically fixed for 4 days or less) in 10% Formalin atroom temperature, then briefly washed in PBS and transferred and keptinto 70% Ethanol (Fisher Scientifics) prior to being transferred to thePathology Translational Research Core at the Geisel School of Medicineat Dartmouth where they were paraffin embedded, sectioned and thenstained.

Paraffin embedded tissue sections (4 μm) were stained using a Leica BONDRX automated stainer. After dewaxing, the sections were subjected toantigen retrieval (Bond epitope retrieval solution 2, 100° C., 20minutes) and incubated with the primary antibody (see dilution in Table17, below) for 30-60 minutes, at room temperature in Leica diluent.Slides were then washed 3×5 min washes in PBS and incubated withsecondary antibody (from Leica Bond Refine detection kit, DS9800). After3 final washes in PBS the sections were incubated with DAB (Leica Bondpolymer detection kit), rinsed, counterstained with hematoxylin andmounted.

Slides were scanned with a Leica (Aperio® AT2, SCN400) whole slidescanner. Whole slide scans were quantified using HALO software (IndicaLabs).

TABLE 17 Antibodies used in staining Specificity Ig type Clone/FormatCatalog # Company Retrieval Dilution mouse CD3 rabbit polyclonal AB5690Abcam EDTA 1:300 mouse CD4 rabbit clone 1 50134-R001 SinoBiologicalsEDTA 1:400 mouse MPO rabbit polyclonal A0398 Dako EDTA 1:1000 mouseCD11b rabbit polyclonal ab-75476 Abcam EDTA 1:300 mouse F4/80 rabbitclone SP115 NBP2-12506 Novus EDTA 1:100

Results

Myeloperoxidase (MPO)-stained slides were scanned as described hereinand evaluated in Aperio® ImageScope. The twelve tumor samples (6 migG2a,6 VSTB123 tumors) were visualized singly and in aggregate. MPO-stainedpositive cells had morphology consistent with neutrophils. InIgG-treated controls, MPO cells showed dense but focal, well-demarcatedclusters located throughout the tumor tissue. Typically, these denseaggregates surrounded a single adipocyte. More MPO-positive cells wereobserved in VSTB123-treated tumors, showing a much broader infiltrativedistribution into the tumor parenchyma compared to mIgG2a controls.

Example 29: VSTB174 Induces Transient Neutrophil Decrease in Blood

VSTB174 (CNTO 8548) was administered to cynomolgus monkeys byintravenous bolus injection for 1 month to evaluate potential toxicityand to evaluate the potential reversibility of toxicity, if any.Clinical pathology parameters (e.g., hematology) were measured,including red blood cell mass and while blood cell counts. As shownherein, VSTB174 induced a transient decrease in circulating neutrophils.

Methods

For purposes of this study, procedures described herein apply to allanimals through Day 36.

Test article: VSTB174, at 50 mg/ml, maintained at −70° C. and protectedfrom light. On each day of dosing, new vials of stock test article wereremoved from frozen storage, equilibrated for approximately 1 hour toambient room temperature and the vial was swirled gently (was not shakenor vortexed) to mix the solution until it was homogeneous. The nominalconcentration (50 mg/mL) of the test article was used for dilutioncalculations for preparation of the dose solutions. The formulationswere prepared by diluting the test article with the control article(0.9% Sodium Chloride) while under a biosafety hood. The final testarticle formulation was filtered through a 0.22 micron syringe filter(PVDF membrane) and held for no longer than 4 hours prior to fillingappropriately-sized syringes for dosing. Syringe size was the smallestpossible for the volume to be administered. The prepared dosing syringeswere used within 4 hours of preparation. The preparation procedure wasmaintained in the raw data. Residual volumes were discarded.

Control article: 0.9% sodium chloride for injection, USP; batch numberP326603, stored at room temperature.

The experimental design is shown in Table 18.

TABLE 18 Experimental design Dose No. of Animals Level Dose Dose ManiRecovery Group Test Dose (mg/kg/ Volume Conc. Study Study No. MaterialRoute week) (mL/kg) (mg/mL) Males Females Males Females 1 VSTB174 IV 0 20 3 3 2 2 2 VSTB174 IV 10 5 3 3 2 2 3 VSTB174 IV 30 15 3 3 2 2 4 VSTB174IV 100 50 3 3 2 2

Administration of Test and Control Articles

The test or control articles were administered to the appropriateanimals in Groups 1, 2, 3, and 4 via intravenous (slow bolus) injectioninto a suitable peripheral vein once weekly for 5 weeks (i.e., Days 1,8, 15, 22, and 29) for a total of 5 doses. The dose volume for eachanimal was based on the most recent body weight measurement obtained upto the day prior to dosing. The animals were temporarily restrained fordose administration and were not sedated. Disposable sterile syringeswere used for each animal/dose. The first day of dosing was designatedas Day 1.

Sample Collection

Blood was collected by venipuncture. Urine was collected by drainagefrom special stainless steel cage pans pretreatment and on the day ofnecropsy. When cage pan collection was unsuccessful, urine was collectedby cystocentesis at necropsy. After collection, samples were transferredto the appropriate laboratory for processing. Animals were fasted priorto clinical chemistry blood collections. Samples were collected asfollows: Week (−2), Week (−1), Day 1 (4 hours post dose), Day 2, Day 4,Day 8 (pre), Day 15 (pre), Day 22 (pre), Day 29 (4 hours post dose), Day31, Day 34, Week 6, Week 7, and Week 8.

Hematology

Blood samples were analyzed for neutrophil count (absolute). A bloodsmear was prepared from each hematology sample. Blood smears werelabeled, stained, stored, and archived.

Results

Generally, administration of VSTB174 by once-weekly intravenous (slowbolus) injection for 5 weeks (i.e., Days 1, 8, 15, 22, and 29) for atotal of 5 doses was generally well tolerated in cynomolgus monkeys atlevels <30 mg/kg/week Neutrophils were markedly decreased beginning onDay 2 with a gradual return to baseline by Day 29 followed by post-dosedecreases at 100 mg/kg/week on Days 31 and 34 (FIG. 46).

Example 30: Vstb174 Induces Activation of NK Cells, Monocytes, and TCells

As described herein (e.g., Example 26), VSTB174 induces significantactivation of monocytes in whole PMBC cultures as indicated by cellsurface markers and cytokine production. The present study was designedto determine the time course for activation of monocytes, T cells and NKcells across three unique donors. Correlative expression of cytokineswas also analyzed for comparison to the in vivo mouse studies describedin Example 26. VSTB140 was also tested to determine the contribution ofFcR binding.

Methods

Preparation of Media

500 ml of RPMI 1640 (Corning, 10-040-CV) were combined with 50 ml ofhuman AB serum (Valley Biomedical, Inc, Lot #3C0405), 5 ml ofPenicillin/Streptomycin (Lonza, 17-602E) 10,000 U/ml, 5 ml ofL-glutamine (100×) (Gibco, 25030-081) and 10 ml of HEPES (1M) (FisherBP299-100, Lot #-1). Media was stored for no longer than 14 days at 4°C.

Preparation of Anti-VISTA and Control Antibodies

Antibodies (VSTB174 or VSTB140) were diluted to 2× desired concentrationin 10% AB serum media. Added 100 μl of the appropriate antibodysolutions to the appropriate wells of a 96 well U-bottom plate (Falcon,353077). After the cells were added in 100 μl, the final concentrationof each antibody was 10, 1, 0.1 or 0.01 μg/ml. IgG1 control antibodyCNTO 3930 (Lot 6405, ENDO<0.1 EU/mg) or IgG2σ control antibody CNTO 8937(Lot 7421, ENDO<0.1 EU/mg) was added at a final concentration of 10μg/ml. Each condition was run in triplicate.

Isolation of PBMC

Donors were at least 18 years of age, generally healthy and selectedrandomly from the local population. Three donors provided PBMCs for thisstudy. Donor blood was transferred from isolation tube to 50 ml conicalsand under-laid with 15 mls of Ficoll 1077 (SIGMA, 10771) being carefulnot to mix with the blood. This was per 25 mls of blood. The cells werecentrifuged at 1250 g for 25 minutes at room temperature with no brake.White blood cells were isolated at the interphase of the Ficoll and theserum and diluted the cells into 40 ml of Hanks Balanced Salt Solution(HBSS). Cells were centrifuged at 453 g (1500 rpm) for 10 minutes at 4°C. Cells were resuspended in 50 mls of HBSS and counted by transferring500 ?alto a separate eppendorf tube.

In Vitro Culture Setup

The appropriate number of cells needed for the assay was determinedbased on the number of samples to be analyzed. The PBMCs were seeded at2.0×10⁵cells/well of a 96 well U-bottom plate. All conditions wereperformed in technical triplicates. Cells were centrifuged T 453G (1500rpm) for 10 minutes at 4° C., and resuspended at a concentration of2×10⁶/ml in 10% AB serum media and added 100 μl to appropriate wellsbringing the total volume in each well to 200 μl. Cells were incubatedfor 24 hours at 37° C. and 5% CO₂. Collected 100 μl of supernatant foranalysis by Luminex.

Multiplex analysis was carried out according to the method described inExample 26.

Antibody Staining and Flow Cytometry

The 96 well U-bottom plate was centrifuged for 5 minutes at 453 g andremoved the supernatant. Cells were washed with 200 μl PBS andcentrifuged again under the same conditions. Supernatant was discardedand cells were resuspended in 50 μl of PBS containing the followingantibodies:

Monocytes

-   -   CD14-APC (clone HCD14) 1:250 (Biolegend cat #325608)    -   HLA-DR-PE Cy7 (clone L243) 1:250 (Biolegend cat #307616)    -   CD80-PE (clone 2D10) 1:250 (Biolegend cat #305208)    -   Hu FcR binding inhibitor (eBioscience cat #14-9161-73)

NK cells

-   -   CD3 (clone UCHT1) 1:200 (Biolegend cat #300420)    -   CD56 (clone HCD56) 1:200 (Biolegend cat #318327)    -   CD25 (clone M-A251) 1:200 (Biolegend cat #356110)    -   CD69 (clone FN50) 1:200 (Biolegend cat #310906)

T cells

-   -   CD3 (clone SK7) 1:200 (Biolegend cat #344814)    -   CD4 (clone OKT4) 1:200 (Biolegend cat #317414)    -   CD8 (clone SK1) 1:200 (Biolegend cat #344713)    -   CD25 (M-A251) 1:200 (Biolegend cat #356110)    -   CD69 (clone FN50) 1:200 (Biolegend cat #310916)    -   CD62L (clone DREG-56) 1:200 (Biolegend 304827)

Cells were incubated for 20 minutes on wet ice in the dark. 150 μl ofPBS were added and cells were centrifuged for 5 minutes at 453 g. Thecells were washed again with 200 μl of PBS and centrifuged under thesame conditions; 120 μl of PBS were added. Samples were run on aMiltenyi MACSQuant 10-parameter flow cytometer and analyzed using FlowJo9.7.5 for expression of HLA-DR, CD80 and Annexin-V on the CD14+population. Median fluorescence intensity (MFI), a statistic thatdefines the central tendency of a set of numbers, was used as thedefining statistic to compare treatments.

Statistical Analysis

All statistics for the cytokine analysis were carried out in RStatistical Computing Language. Cytokine concentration values belowdetection (<OOR) were rescaled to the lowest detectable concentration,and values above accurate quantitation (>OOR) were rescaled to themaximum linearly quantifiable concentration. Statistical outliers wereremoved prior to statistical analysis on the basis of Grubbs' p<0.05 andoutlier distance of greater than 1 standard deviation from the groupmean using a single step. Pair-wise comparisons amongst the groups weremade at each of the time-points using One-Way ANOVA with Tukey HonestSignificant Differences. P-values less than 0.05 for all tests andcomparisons were deemed significant. Using the heatmap.2 function in theg-plots package in R, complete hierarchical clustering of cytokines wasassessed to generate a heatmap (data not shown).

All statistics for the flow cytometric analysis were carried out inPrism GraphPad, version 6. Pair-wise comparisons amongst the groups weremade at each of the time-points using Two-Way ANOVA with Tukeycorrection for multiplicity for comparing concentrations between groupsand a Dunnett's post-test for comparisons of each group within each timepoint. P-values less than 0.05 for all tests and comparisons were deemedsignificant. For all graphs and tables, *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

Results

Whole PBMCs were isolated from three unique donors and incubated witheither VSTB174 or VSTB140 at a range of concentrations described herein.At 3, 6 and 24 hours, the supernatants were collected and frozen whilethe PBMCs were stained with a range of antibodies for activation markerson monocytes, T cells or NK cells.

NK cell activation was measured by the expression of CD25 and CD69 onthe CD56+CD3-population of cells. At 3 hours, CD69 was slightlyupregulated in the VSTB174 treated group. By 6 hours VSTB174 treatmentenhanced expression of CD69 on NK cells from donors 301 and 302, and toa lesser extent in donor 303. CD25 expression was decreased in theseearly time points for donors 301 and 302 with minimal changes observedfor donor 303. By 24 hours VSTB174 treatment resulted in strongupregulation of both CD69 and CD25 in donors 301 and 302, however donor303 had upregulated CD69, but decreased expression of CD25. A doseresponse of NK cell activation was observed with statisticallysignificant changes between 10 and 0.1 μg/ml. VSTB140 did not inducesignificant upregulation of any markers.

Monocyte activation was measured by the expression of CD80, HLA-DR andPD-L1 on the CD14+ population of cells. At 3 hours, no sign ofactivation was observed for any marker in the three donors with eitherantibody. At 6 hours VSTB174 treatment enhanced expression of PD-L1 onmonocytes from donors 301 and 302, but not in donor 303. CD80 and HLA-DRalso showed small signs of enhanced expression at this time point. By 24hours VSTB174 treatment resulted in upregulation of CD80, HLA-DR andPD-L1 in donors 301 and 302. Donor 303 also upregulated all threemarkers but to a lesser extent. In all cases, a dose response ofmonocyte activation was observed with statistically significant changesbetween 10 and 0.1 μg/ml. VSTB140 did not induce significantupregulation of any markers.

T cell activation was measured by the expression of CD69, CD25 and CD62Lon the CD3+CD4+ and CD3+CD8+ population of T cells. At 3 hours, no signof activation was observed for any marker on either CD4 or CD8 T cells,with the exception of donor 301 where CD69 was modestly upregulated. At6 hours, no consistent activation was observed on the CD4+ population ofT cells, however the CD8+ T cells had increased expression of CD69 fordonors 301 and 302. By 24 hours, both the CD4+ and CD8+ T cellpopulations had increased expression of CD69 and CD25 in donors 301 and302. While donor 303 tended to have elevated levels of CD25 and CD69 onthe CD4+ population, no statistical difference was observed for anyparameter. CD62L expression was not changed at any time point withVSTB174. VSTB140 did not induce any consistent changes in expression ofCD69, CD25 or CD62L at any time point across the donors.

As shown herein, VSTB174, but not VSTB140, induced activation of NKcells, T cells and monocytes. Activation of NK cells was seen as earlyas 3 hours. Monocyte and T cell activation was observed starting at 6hours. For all three populations, maximal activation was observed at the24 hour time point. Representative in vitro induction of monocyte, NK,and T cell activation from donor 302 at 24 hours post VSTB174 treatmentis shown in FIG. 48. Further, VSTB174, but not VSTB140, induced enhancedproduction of multiple cytokines and chemokines from PBMCs in multiplehuman donor samples, in a dose-dependent fashion. Cytokine productionwas observed as early as 3 hours, with the largest fold changes observedat 24 hours (data not shown).

Example 31: Macrophages Contribute to Anti-Vista Mechanism of Action inthe Mb49 Tumor Model

The present study was conducted to determine whether macrophages,monocytes and/or granulocytes play a role in MB49 tumor growth controlmediated by an anti-hVISTA antibody, VSTB123, in female hVISTA KI mice.

Methods

Study Design

The hVISTA KI female mice were divided into 8 treatment groups (Table19). Each mouse was injected with MB49 tumor cells in the right flank onday 0. At days 7, 9, and 11, mice were treated by ip injection with 10mg/kg of mIgG2a control antibody or VSTB123. Groups 1 and 2 served assimple negative and positive controls to demonstrate the therapeuticeffect of VSTB123, with no additional treatment to deplete myeloidsubsets. Groups 3 and 4 received control rIgG2b and groups 7 and 8received anti-GR1 antibody on days 5, 7, 9, 11, 13, 15 and 17 post tumorinjection. Groups 3 and 4 received control liposomes and groups 5 and 6received clodronate liposomes on days 4, 10, and 16. Blood from mice wasanalyzed on days 7 and 22 to confirm immune cell depletion. Tumors weremeasured 2 times per week for four weeks to monitor efficacy. Survivalwas monitored to day 78. FIG. 49 shows a schematic of the study design.

TABLE 19 Study design summary Group Treatment N (early deaths) 1(negative control for mIgG2a 8 VSTB123) 2 (positive control) VSTB123 9 3(negative controls for both mIgG2a, rIgG2b, 8 (1 mouse died depletionmodalities and for control liposome early) VSTB123) 4 (negative controlfor both VSTB123, rIgG2b, 9 depletion modalities; with control liposomeVSTB123 treatment) 5 (macrophage/DC depletion mIgG2a, clodronate 14 (6died due to and negative control mAb for liposome liposome toxicity)VSTB123) 6 (macrophage/DC depletion VSTB123, 14 (7 died due to andVSTB123) clodronate liposome liposome toxicity) 7 (granulocyte/monocytemIgG2a, anti-GR1 10 depletion and negative control for VSTB123) 8(granulocyte/monocyte VSTB123, anti-GR1 10 depletion and VSTB123)

Mice

hVISTA KI mice have the human VISTA cDNA sequence inserted into theexisting mouse VISTA locus in place of the mouse VISTA gene. The miceexpress only human VISTA RNA and protein. Breeding and mouse husbandrywas contracted to Sage Labs (PA, USA). Prior to use, all mice incomingfrom Sage were quarantined for 3 weeks and screened against infection.After clearing quarantine, all mice were transferred to the regularfacility. The mice were acclimated for a minimum of 2 days prior tohaving their flanks shaved, their tails tattooed, and tumor cellsinjected.

Tumors

The MB49 cells were confirmed to be free of mycoplasma and othercontaminants (IMPACT SC testing at IDDEX RADIL Case #22209-2014). Onecell vial was thawed and grown in RPMI 1640 (+L-Glut) with 10% FBS,non-essential amino acids and pen/strep antibiotics. After three days inculture, cells were harvested by brief incubation with StemPro Accutase,washed twice and resuspended in cold RPMI at a concentration of 4.0×10⁶cells/ml, and 50 μl (2×10⁵ cells) injected per mouse. All culturereagents were purchased from Gibco and Hyclone. Mice were injectedintradermally in their shaved flank with 50 μl of MB49 cell suspension(˜200,000 cells).

Test Agents, Depleting Antibodies, and Clodronate Liposomes

VSTB123 is an anti-human VISTA antibody comprised of the VSTB174variable region on a muIgG2a Fc scaffold. Control mouse antibody(mIgG2a) was generated by BioXcell (clone C1.18.4, lot #5386-2/1014).Mice were dosed with VSTB123 or mIgG2a isotype control at 10 mg/kg.

Anti-GR1 antibody was generated by BioXcell (clone RB6-8C5, lot#5246/1114; rat IgG2b) and depletes monocytes, granulocytes, and MDSCexpressing GR1 antigen. Control rat IgG2b was procured from BioXcell(clone LTF-2, lot #5535-3-6-7/0515). Mice were dosed with anti-GR1 orrIgG2b isotype control at 12.5 mg/kg.

Clodronate liposomes or control liposomes were purchased fromClodronateLiposomes.com (lot #3574E). Mice were dosed with 200 μgclodronate liposomes or control liposomes, diluted in PBS.

Tumor Measurement

Tumor growth was evaluated two times per week for four weeks (day 38).The formula (L×W²)/2 was used to determine tumor volume (L is the lengthand W the width of the tumor. As per Dartmouth's IACUC requirements,animals were euthanized as soon as their tumors reached 15 mm in thelongest dimension or showed any sign of distress or weight loss over 20%of their normal body weight. Mouse deaths were recorded throughout theexperiment.

Partial regression (PR) was reached when tumor was half (or more reducedin size but greater than 13.5 mm³) of the initial volume for 3consecutive measurements. Complete regression was reached when any tumorwas less than 13.5 mm³ for 3 consecutive measurements. Mice that werealive on day 38 were observed for survival to day 78.

Blood Recovery and Flow Cytometry

Mice were bled from the retro-orbital cavity on days 7 and 22 to confirmthe depleting effects of anti-GR1 or clodronate treatments, usingPasteur pipettes dipped in heparin. Five mice were bled per group andblood was analyzed by flow cytometry. Blood was subjected to red bloodcell lysis in 3 ml ACK lysis buffer (Lonza, Lot No. 0000400419) for 5minutes. After 1 wash in HBSS, cells were resuspended in PBS and usedfor immunostaining.

Single-cell suspensions were Fc-blocked with anti-murine CD16/32(Miltenyi) (1:200) for 15 minutes at 4° C. Cells were incubated withantibody cocktails diluted in flow buffer (PBS with 5 mM EDTA and 0.5%w/v BSA) for 30 min.

Panel:

-   -   Live/Dead Yellow (Life Technologies) (1:1000)    -   Ly6G-FITC (Biolegend, 1A8) (1:200)    -   CD45-PE (Biolegend, 30-F11) (1:800)    -   Ly6C-PerCP/Cy5.5 (Biolegend, HK1.4) (1:100),    -   CD11b PE/Cy7 (Biolegend, M1/70)(1/200),    -   F4/80-APC/Cy7 (Biolegend, BM8) (1/200),

After 2 washes in PBS, cells were resuspended in flow buffer and run ona MACS Quant flow cytometer. All cells were gated on live/dead andpositive CD45 staining. Granulocytes were CD11b+, Ly6G+ and Ly6C−.Monocytes were CD11b+, Ly6G−, and Ly6C+. Macrophages were CD11b+, Ly6G−,Ly6C−, and F4/80+. The CD11c marker was not included so effects ofclodronate liposomes on dendritic cells cannot be determined. Flowcytometry data was analyzed using Flow Jo v9.

Statistical Analysis

Statistical analysis of tumor growth rates were conducted using R 3.0and a macro, inference.R. Survival curves were compared using Log-rank(Mantel Cox) analysis from Prism v6.0. p values <0.05 were consideredsignificant.

Results

The depletion regimens were monitored on day 7 and 22 for impact onrelevant circulating blood populations. Macrophages as a percentage ofCD45+ cells were reduced by about 40% with clodronate liposometreatment, when measured on day 7, three days after depletion. Since thedepletion was less than complete and/or not sustained, the contributionof macrophages (and dendritic cells) to VSTB123-mediated anti-tumorefficacy may be underestimated in the results.

Anti-GR1 treatment reduced monocytes by ˜75% and granulocytes by ˜98%two days after depletion started on day 5. It is possible that thistreatment would underestimate somewhat the effect of monocytes inVSTB123-mediated anti-tumor efficacy, though granulocytes werewell-depleted. All populations recovered by day 22, 6-7 days after thelast depletion treatment.

As a baseline, the efficacy of VSTB123 was compared to isotype controlantibody in the absence of any myeloid depleting treatments. MB49-tumorbearing female hVISTA KI mice dosed with VSTB123 had significantlyreduced tumor burden compared to control mIgG2a treated animals(interaction P value=0.000177; data not shown). In addition, on day 31there were 5/9 CR and 3/9 PR in the VSTB123 group, while there was just1/8 PR in the mIgG2a group.

To determine if liposome vehicle and isotype rat control antibody had aneffect on tumor burden, animals treated with these controls plus eithermIgG2a or VSTB123 were compared to animals treated with mIgG2a orVSTB123 alone. The negative control treatments did not affect tumorvolume of mice treated with mIgG2a (interaction p value=0.306; data notshown). However, VSTB123 was significantly less efficacious in thepresence of control liposomes/rIgG2b (interaction P-value=0.049). Thenegative effect of the control agents on VSTB123 efficacy is supportedby reduced incidence of CR (3/9) and PR (2/9) in this group, as comparedto mice treated only with VSTB123. This suggests that the controlliposomes and/or rat control antibody had an adverse effect on efficacymediated by VSTB123, more likely the control liposomes.

Clodronate liposome treatment, which depletes macrophages and dendriticcells, eliminated the anti-tumor effect of VSTB123 (FIG. 50, left panel;interaction P value=0.100 for mIgG2a/clodronate liposomes vsVSTB123/clodronate liposomes). However, this may be an underestimate ofthe importance of macrophages and dendritic cells, as depletion usingclodronate liposomes was not complete or sustained (data not shown).This result is consistent with a reduced incidence of CR (0/7) and PR(1/7) on day 31 in the VSTB123/clodronate liposome mice. Clodronate andcontrol liposome mIgG2a treatment groups showed significant interactionp values with the mouse IgG2a group, but both groups showed aggressivetumor growth similar to mIgG2a (FIG. 50, left panel). This resultsuggests that macrophages/DC do not play a prominent role in controllingor promoting tumor growth in the natural MB49 tumor model. However, theresults of clodronate liposome treatment in VSTB123-treated mice suggestthat anti-VISTA treatment may enhance macrophage/DC activity as amechanism to control MB49 tumor growth.

The anti-GR1 antibody is expected to deplete monocytes and granulocytes,and myeloid derived suppressor cells (MDSC); the latter cell type isexpected to support tumor growth. Substantial depletion of monocytes andgranulocytes was observed after one dose of anti-GR1 antibody (data notshown). Treatment of VSTB123 mice with anti-GR1 antibody did notadversely affect efficacy mediated by the anti-VISTA antibody, and thetumor volume suggests a non-significant positive impact. The incidenceof CR (7/10) and PR (2/10) in the GR1NSTB123 group is somewhat higherthan in the VSTB123 group alone. In addition, depletion ofmonocytes/granulocytes significantly reduced tumor growth in the mIgG2acontrol group (data not shown). The increased incidence of CR (2/10) andPR (2/10) in the GR1 depleted mIgG2a group as compared to mIgG2a aloneis further support for a positive impact of the GR1 depleting antibody.The results suggest that monocytes/granulocytes/MDSC promote MB49 tumorgrowth, but these cells do not seem to be affected by VSTB123 treatment,or part of the anti-tumor mechanism induced by VSTB123. The incidence ofcomplete regression (CR) and partial regression (PR) is summarized inTable 20.

TABLE 20 Incidence of CR and PR in the treatment groups at day 31.mIgG2a, VSTB123, mIgG2a, VSTB123, ctrl lipo, ctrl lipo, clodronateclodronate mIgG2a, VSTB123, mIgG2a VSTB123 rIgG2b rIgG2b liposomesliposomes anti-GR1 anti-GR1 CR 0/8 5/9 1/7 3/9 1/8 0/7 2/10 7/10 PR 1/83/9 0/7 2/9 0/8 1/7 2/10 2/10

Survival analysis was performed on MB49-tumor bearing animals treatedwith mIgG2a or VSTB123 in the presence or absence of depleting GR1antibody or clodronate liposomes. The survival curves generated for thepresent analysis only included animals that died as a direct result oftumor burden. Animals that died from clodronate treatment or who had tobe euthanized early due to veterinarian's instructions have been removedfrom the analysis.

Survival curves for mIgG2a and VSTB123 were significantly different(P=0.0279; data not shown), consistent with the significant reductionsin tumor burden achieved with VSTB123. Survival curves for mIgG2a andVSTB123 in the presence of control liposome and rat isotype controlantibody were not found to be different (data not shown), suggesting anegative effect of the control PBS-loaded liposome treatments on VSTB123control of tumor growth.

Survival curves between mIgG2a and VSTB123 in the presence of clodronateliposomes were also not found to be different (data not shown),suggesting clodronate liposomes were negatively affectingVSTB123-mediated efficacy. In addition, survival was significantlydifferent when comparing VSTB123 vs VSTB123/clodronate liposomes(increased survival with VSTB123 alone; data not shown). This resultindicates that depletion of macrophages/dendritic cells had asignificant impact on the anti-tumor response induced by VSTB123.

The effect of depleting monocytes/granulocytes/MDSC on survival was alsoassessed. Survival was significantly increased in mice treated withVSTB123/anti-GR1 as compared with mIgG2a/anti-GR1 (p=0.0055; data notshown). Survival approached significance for mIgG2a vs mIgG2a/anti-GR1(p=0.0880), suggesting that the depletion of monocytes/granulocytes/MDSChad a beneficial effect on survival in the MB49 tumor model, independentof VSTB123 treatment. Survival was not significantly different in micetreated with VSTB123 vs VSTB123/anti-GR1 (p=0.4368). The resultsindicate that monocytes/granulocytes/MDSC do not play a significant rolein anti-tumor efficacy mediated by VSTB123.

To summarize, VSTB123 treatment significantly reduced tumor volume andprolonged survival when compared to mice treated with migG2a controlantibody. Monocytes, granulocytes, and MDSC were depleted in mice usingan anti-GR1 antibody. Depletion of these cells did not significantlyaffect VSTB123-mediated efficacy as measured by tumor volume andsurvival, indicating that these cells do not contribute to anti-VISTA.mechanism of action in the MB49 tumor model. Macrophages were depletedin anti-VISTA treated mice using clodronate liposomes. Macrophagedepletion significantly reduced VSTB123-mediated efficacy as measured bytumor volume and survival, indicating that macrophages contribute toanti-VISTA mechanism of action in the MB49 tumor model.

Example 32: Cd4+ and Cd8+ T Cells Contribute to Anti-Vista Mechanism ofAction in the Mb49 Tumor Model

The present study was conducted to determine the efficacy of VSTB123 inthe MB49 tumor model in female hVISTA KI mice depleted of CD4+ T cells,CD8+ T cells, or NK cells. As described herein, this knock-in mouse linehas the human VISTA cDNA knocked-in in place of the mouse VISTA gene,and expresses only human VISTA both at RNA and protein level. MB49 tumorcells express male H-Y antigen, a self-antigen in male mice but aforeign antigen in female mice.

Mice were injected with MB49 cells and then depleted of lymphocytesubsets prior to the initiation of VSTB123 treatment. The mice received3 doses of VSTB123 or control antibodies at 10 mg/Kg every other day forthree doses. Cell subset targeted depletion was done through the use ofdepleting antibodies against cell surface markers CD4 (clone GK1.5), CD8(clone 2.43), or NK1.1 (clone PK136). Tumor volume and survival weremonitored.

Methods

hVISTA KI mice were divided into 8 groups as indicated in Table 21.Groups 1 and 2 were treated with VSTB123 or control mIgG2a antibodies,and served as the baseline controls for tumor growth with and withoutanti-VISTA treatment. Both the anti-CD4 (GK1.5) and anti-CD8 (2.43)depleting antibodies are of the rat IgG2b isotype, so groups 1 and 2were dosed with rIgG2b isotype control for the depleting antibodies. TheNK1.1 antibody is a mouse IgG2a, so the mIgG2a control antibody forVSTB123 served as a control for NK depletion. Groups 3 and 4 weredepleted of CD4+ T cells, while groups 5 and 6 were depleted of CD8+ Tcells. Anti-NK1.1 was used to deplete NK cells in groups 7 and 8.

TABLE 21 Treatment groups - following MB49 cell injection, mice wererandomized into 8 groups of 10 mice per group and treated with theindicated antibodies. Group Treatment N 1 Control mIgG2a, rat IgG2b 10 2VSTB123, mIgG2a, rIgG2b 10 3 mIgG2a, aCD4 (GK1.5) 10 4 VSTB123, aCD4(GK1.5) 10 5 mIgG2a, aCD8 (2.43) 10 6 VSTB123, aCD8 (2.43) 10 7 mIgG2a,aNK1.1 (PK136) 10 8 VSTB123, aNK1.1 (PK136) 10

Five days after MB49 tumor cell implantation in hVISTA KI female mice,initiation of the depletion regimen began. Depleting antibodies wereadministered on day 5, 2 days prior to the first VSTB123 or mIgG2atreatment on day 7, and then throughout the study (FIG. 51). Tumormeasurements were made 2 times a week for four weeks. Each group of micewas treated with 3 doses of VSTB123 or mIgG2a control antibody at 10mg/Kg on days 7, 9 and 11. See FIG. 51 for experimental design.

Cell Source and Preparation

The MB49 cells were confirmed to be free of mycoplasma and othercontaminants (IMPACT SC testing at IDDEX RADIL Case #22209-2014). Onecell vial was thawed and grown in RPMI 1640 (+L-Glut) with 10% FBS andpen/strep antibiotics. After three days in culture, cells were harvestedby brief incubation with StemPro Accutase, washed twice and resuspendedin cold RPMI at 4×10⁶ cells/ml prior to injection into the mice. Allculture reagents were purchased from Gibco and Hyclone.

Test Agents and Dosage

As described herein, VSTB123 is a chimeric anti-human VISTA antibodythat contains the anti-human VISTA variable region derived from VSTB174,cloned into a mouse IgG2a Fc backbone. VSTB123 and mIgG2a (BioXcellBE0085, clone C1.18.14, lot #5386-2/1014) were diluted in PBS andinjected via intraperitoneal route in a volume of 0.2 ml to deliver adose of 10 mg/kg. Animals received a total of 3 doses injected everyother day. For depletion of lymphoid cells, mice were treated withanti-CD4 (clone GK1.5; BioXCell; lot #4912/0114), anti-CD8 (clone 2.43;BioXCell; lot #4933/1213), or anti-NK1.1 (clone pk136; BioXCell; lot#4945/0114) as indicated in FIG. 51. Depleting antibodies were dilutedin PBS to a concentration of 1.25 mg/ml and injected via intraperitonealroute in a volume of 0.2 ml to deliver 250 μg/mouse.

Mice

The hVISTA KI female mice are bred at Sage Labs (Boyertown, Pa.). Themice, aged 8-12 weeks, first transited for 3 weeks in the quarantinefacility, and then were transferred to the regular facility. They wereacclimated for 2 days prior to having their right flanks shaved andtheir tails tattooed. Tumor cells were injected 2 days later.

Intradermal Cell Injection and Randomization

Mice were injected intradermally in their shaved right flank with 50 μlof MB49 cell suspension (˜200,000 cells). All mice in which theinjection went poorly (leak from injection site or subcutaneousinjection instead of intradermal) were removed from the experiment. Micewere randomized into eight groups on day 4.

Treatment Initiation and Tumor Measurement

On day 5 after tumor cell injection, cell subset depletion was initiatedvia administration of depleting antibodies against the surface markersCD4, CD8, or NK1.1. VSTB123 or control antibody treatment was initiatedon day 7 post-MB49 cell injection. The animals received 3 VSTB123 ormIgG2a doses and tumor growth was monitored 2 times a week for 4 weeksfollowing VSTB123/mIgG2a treatment. Tumors were measured starting on day7 post-MB49 cell-injection. The formula (L×W²)/2 was used to determinetumor volume (L is the length or longest dimension, and W is the widthof the tumor).

Partial regression (PR) was reached when tumor was half (or more reducedin size but greater than 13.5 mm³) of the initial volume for 3consecutive measurements. Complete regression was reached when any tumorwas less than 13.5 mm³ for 3 consecutive measurements.

Euthanasia Criteria

As per Dartmouth's IACUC requirements, animals were euthanized as soonas their tumors reached 15 mm in the longest dimension or showed anysign of distress or weight loss over 20% of their normal body weight.Mouse deaths were recorded throughout the experiment.

Blood Collection and Flow Cytometry Analysis

Mice were bled from the retro-orbital cavity 4 days prior to MB49 cellimplantation and again on day 7-post cell implantation to confirm thedepleting effects of anti-CD4, anti-CD8, and anti-NK1.1 antibodies viaflow cytometry. Blood was placed into Eppendorf tubes containing 25 μlof heparin and stained with the following cocktail of antibodies in FACsbuffer (2% BSA, 1 mM EDTA, 0.02% sodium azide):

-   -   αCD335 (NKp46) (clone 29A1.4)−BV421 (1:200)    -   αCD3—FITC (1:200)    -   αCD45—PE (1:800)    -   αCD4 (clone RM4-5)—PerCP-Cy5.5 (1:400)    -   αCD8 (clone 53-6.7)—APC (1:400)    -   Fc block—1:200

Following staining with antibody clones that bind to noncompetingepitopes of the depleting antibodies, blood was subjected to red bloodcell lysis in ACK lysis buffer (Lonza) for 5 minutes. After 2 washes inFACs buffer, cells were resuspended in PBS and run on a MACSQuantcytometer. Flow cytometry data was analyzed with FlowJo version 9.5.

Statistical Analysis

Mouse tumor volumes were analyzed using Excel for data management andGraphPad Prism for graphing. Statistical analysis was performed using amacro for R statistical computing software that measures divergence intumor volume between two groups of differentially treated mice and isnamed ‘mixed effect repeated measures’. Significance was reached wheneither the main p value or the interaction p value was less than 0.05.Survival analysis was performed in GraphPad Prism using the Log-rank(Mantel-Cox) test. Significance was reached when the p value was lessthan 0.05.

Results

CD8+ and CD4+ T cells were necessary for the anti-tumor response inducedby VSTB123 in the MB49 model (FIG. 50, middle and right panels).Depletion of CD8+ or CD4+ T cells abrogated VSTB123-mediated effects ontumor volume and survival. In mice treated with mIgG2a antibody, onlythe depletion of CD8+ T cells affected tumor growth and survival. Thisindicates that VSTB123 treatment promoted a broader immune response thatincluded CD4+ T cells, as well as CD8+ T cells. Depletion of NK cellsdid not affect VSTB123-mediated efficacy as measured by tumor volume orsurvival. Depletion of NK cells significantly reduced tumor volume inthe mIgG2a group, but this did not translate into a survival benefit.The incidence of complete or partial remission in the treatment groupsare shown in Table 22.

TABLE 22 Incidence of CR and PR in the treatment groups at day 31.mIgG2a VSTB123 mIgG2a VSTB123 mIgG2a VSTB123 mIgG2a VSTB123 CD4 dep CD4dep CD8 dep CD8 dep NK dep NK dep CR 1/10 6/10 0/10 0/10 0/10 0/10 3/105/10 PR 1/10 1/10 1/10 1/10 0/10 0/10 0/10 1/10

Example 33: Vstb123 Demonstrates Synergy with Anti-Pd1 Antibody In Vivo

The present study examined the effectiveness of an anti-VISTA antibody(VSTB123) combined with an anti-PD1 antibody in treating MB49 tumors inmale mice.

Method

Mice

VISTA KI male mice (n=132 to have 3 extra mice per group). Mice wereborn between 7/13-27/15 and 6/15-29/15 and were randomized between thedifferent groups.

Tumors

Each mouse received 250,000 MB49 tumor cells, injected intradermally inthe right flank.

TABLE 23 Treatment groups Group Treatment N 1 mouse IgG2a and rat IgG2a(2A3) 30 2 Anti-VISTA (VSTB123) and rat IgG2a 30 (2A3) 3 anti-PD1(RPMI-14) and mouse IgG2a 30 4 Anti-VISTA (VSTB123) and anti-PD1 30(RPMI-14)

Antibody Treatments

Both antibodies (VSTB123 and anti-PD1) and controls were dosed at 10mg/kg. Treatment began on day 6 post-implantation. Anti-PD1(RMP1-14—BioXcell catalog #BE0146) and rat IgG2a were dosed 2× per week.Anti-VISTA and mouse IgG2a were dosed 3× per week and treatmentproceeded for 3 weeks. FIG. 52 shows a schematic of the treatmentschedule.

Efficacy Study

Tumor volume was measured 2-3 times per week for a period of 4 weeks.Mice whose tumors reached 15 mm in the longest direction were euthanizedin accordance with IACUC requirements. Mice that displayed significantsigns of distress (hunched posture, unresponsiveness, etc.) wereeuthanized at the veterinarian's discretion.

Survival Analysis

All deaths were recorded and annotated for the cause of death (miceeuthanized due to tumor burden vs. death due to sickness/distress) forsurvival analysis. When possible, the lungs of the animals to beeuthanized were collected, mets counted and then processed for paraffinembedding.

Early Cytokine Response

Mice were bled at 6 hours post 1^(st) and 3^(rd) dosage of VSTB123(1^(st) and 2^(nd) anti-PD1 dosage) and plasma was stored at −80° C. forcytokine analysis on a Luminex mouse 32-plex (Eotaxin, G-CSF, GM-CSF,IFN-γ, IL-10, IL-12 (p40), IL-12 (p′70), IL-13, IL-15, IL-17, IL-1α,IL-1β, IL-2, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IP-10, KC, LIF, LIX,M-CSF, MCP-1, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNF-α, VEGF).

Mechanism of Action Studies

At 24 h post 1 dose and 3 doses, 10 mice were sacrificed per group:

-   -   5 for flow cytometry on tumor and on terminal cardiac blood    -   5 for paraffin and frozen embedding of tumors lung and liver),        and ICS on cardiac blood

Flow Panel for Blood Analyses

T-Cell Panel Myeloid Panel CD25 4-1BBL CD3 CD11b CD4 CD11c CD44 CD45CD45 CD80 CD62L CD86 CD69 F4/80 CD8 Ly6C FoxP3 Ly6G Live/Dead Live/Dead

Flow Panel for Tumor Analyses

T-Cell Panel Myeloid Panel CD11c 4-1BBL CD25 CD11b CD3 CD11c CD4 CD45CD45 CD80 CD69 F4/80 CD8 MHC class II FoxP3 Ly6C Ki67 Ly6G Live/DeadLive/Dead

Intracellular Cytokine Staining Analysis

Collected blood cells were stimulated with MB49 cell lysate orPMA/ionomycin for 4-6 hours in the presence of Brefeldin A. Followingstimulation, cells were stained for cell surface antigens and live/deaddye, fixed and RBCs lysed. Cells were then be permeabilized and stainedfor intracellular cytokines. ICS flow panel was as follows: Live/Dead,CD45, CD3, CD4, CD8, IL-2, IFNγ, TNFα.

Results

As shown in FIG. 53, the combined effects of anti-VISTA and anti-PD-1 ontumor growth and survival were synergistic when compared to the effectsof either antibody alone.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-31. (canceled)
 32. A method of promoting T cell immunity in a subjectin need thereof comprising administering: a) an antibody or antibodyfragment thereof comprising an antigen binding region that binds to aV-domain Ig Suppressor of T cell Activation (VISTA) comprising sequenceof SEQ ID NO:25, a VH CDR2 having the amino acid sequence of SEQ IDNO:26 and a VH CDR3 having the amino acid sequence of SEQ ID NO:27, andwhich further comprises an antibody VL domain comprising a VL CDR1having the amino acid sequence of SEQ ID NO:28, a VL CDR2 having theamino acid sequence of SEQ ID NO:29 and a VL CDR3 having the amino acidsequence of SEQ ID NO:30; and b) a PD-1 antagonist antibody or antibodyfragment.
 33. The method of claim 32, wherein the antibody or antibodyfragment which binds VISTA comprises a VH domain comprising SEQ ID NO:37and a VL domain comprising SEQ ID NO:44.
 34. The method of claim 32,wherein the antibody or antibody fragment which binds VISTA comprises anantibody heavy chain comprising SEQ ID NO:61 and an antibody light chaincomprising SEQ ID NO:56.
 35. The method of claim 32, which enhances Tcell activation.
 36. The method of claim 32, wherein the PD-1 antagonistantibody binds to PD-1, PD-L1 or PD-L2.
 37. The method of claim 32,wherein the PD-1 antagonist antibody is nivolumab, pembrolizumab,tremelimumab, or ipilimumab.
 38. The method of claim 32, wherein thePD-1 antagonist antibody and the VISTA antibody together comprise abispecific antibody.
 39. The method of claim 32, wherein the treatedsubject has a cancer.
 40. The method of claim 32, wherein the treatedsubject has a chronic infectious condition.
 41. The method of claim 39,wherein the cancer is a solid tumor, a leukemia, a lymphoma, amyelodysplastic syndrome or a myeloma.
 42. The method of claim 41,wherein the solid tumor is a lung tumor, bladder tumor or breast tumor.43. The method of claim 32, wherein the antibody or antibody fragmentthereof in a) and the antibody or antibody fragment thereof in b) areadministered simultaneously.
 44. The method of claim 32, wherein theantibody or antibody fragment thereof in a) and the antibody or antibodyfragment thereof in b) are administered in sequentially.
 45. The methodof claim 32, wherein the antibody or antibody fragment thereof in a) andthe antibody or antibody fragment thereof in b) are administered in thesame formulation.
 46. The method of claim 32, wherein the antibody orantibody fragment thereof in a) and the antibody or antibody fragmentthereof in b) are administered in separate formulations.
 47. The methodof claim 32, wherein the antibody or antibody fragment thereof in a) andthe antibody or antibody fragment thereof in b) are administeredintravenously.
 48. A pharmaceutical composition comprising: a a) anantibody or antibody fragment thereof comprising an antigen bindingregion that binds to a V-domain Ig Suppressor of T cell Activation(VISTA) comprising sequence of SEQ ID NO:25, a VH CDR2 having the aminoacid sequence of SEQ ID NO:26 and a VH CDR3 having the amino acidsequence of SEQ ID NO:27, and which further comprises an antibody VLdomain comprising a VL CDR1 having the amino acid sequence of SEQ IDNO:28, a VL CDR2 having the amino acid sequence of SEQ ID NO:29 and a VLCDR3 having the amino acid sequence of SEQ ID NO:30; and b) a PD-1antagonist antibody or antibody fragment; and c) a pharmaceuticallyacceptable carrier, diluent, or excipient.
 49. The pharmaceuticalcomposition of claim 48, wherein the antibody or antibody fragmentthereof in a) comprises a VH domain comprising SEQ ID NO:37 and a VLdomain comprising SEQ ID NO:44.
 50. The pharmaceutical composition ofclaim 48, wherein the antibody or antibody fragment thereof in a)comprises an antibody heavy chain comprising SEQ ID NO:61 and anantibody light chain comprising SEQ ID NO:56.
 51. The pharmaceuticalcomposition of claim 48, wherein the antibody or antibody fragment in b)is nivolumab or pembrolizumab.